ES2665793T3 - Light chain antibodies modified by genetic engineering with histidine and genetically modified rodents for their generation - Google Patents

Abstract

A genetically modified rodent comprising in its germ line an immunoglobulin light chain locus comprising (i) a single rearranged gene region of human immunoglobulin light chain variable region comprising human VL and JL segment sequences and (ii) a immunoglobulin light chain constant region gene sequence in operative binding to the single human immunoglobulin light chain variable region rearranged gene sequence, in which the only human immunoglobulin light chain variable region rearranged gene sequence comprises at least one Genetically modified insertion or replacement of a histidine codon that is not encoded by corresponding human germline VL and JL gene sequences, in which the gene sequence of rearranged variable region human immunoglobulin light chain alone encodes an immunoglobulin chain that understand a domi variable light chain nio, in which the histidine codon is in the nucleotide sequence encoding a complementarity determining region (CDR) of the light chain variable domain, in which the light chain variable domain comprises at least one histidine residue at an amino acid position encoded by the at least one inserted or substituted histidine codon, and in which the immunoglobulin light chain encoded by the immunoglobulin light chain locus is capable of pairing with a plurality of heavy chains of immunoglobulin selected by the rodent, in which each of the plurality of immunoglobulin heavy chains specifically bind different epitopes when paired with the immunoglobulin light chain.

Description

Light chain antibodies modified by genetic engineering with histidine and genetically modified rodents for their generation

 5

Cross reference to related requests

The present application claims the benefit under 35 U.S.C. §119 (e) of the US Provisional Application. No. 61 / 611,950, filed on March 16, 2012 and the US Provisional Application. No. 61 / 736,930, filed on December 13, 2012. 10

Field of the Invention

A genetically modified non-human animal (eg, rodent, for example, mouse or rat) that expresses antibodies capable of binding to an antigen in a pH dependent manner is described herein. A method is described herein to make modifications to the sequence of the variable region of the immunoglobulin light chain of a non-human animal, in which the modifications include mutagenesis of residues within the gene of the variable region of the light chain, for example, nucleotides encoding one or more amino acids within a complementarity determining region (CDR), to facilitate in vivo expression of antibodies comprising light chain domains 20 that exhibit binding to pH dependent antigens. Methods for producing pH-dependent antigen-binding antibodies are also described herein.

Background of the invention

 25

Antibodies typically comprise a homodimeric heavy chain component, in which each heavy chain monomer is associated with an identical light chain. Antibodies having a heterodimeric heavy chain component (for example, bispecific antibodies) are desirable as therapeutic antibodies. But the preparation of bispecific antibodies having a suitable light chain component that can be satisfactorily associated with each of the heavy chains of a bispecific antibody 30 has proved problematic.

In one approach, a light chain could be selected by probing usage statistics for all variable domains of the light chain, the identification of the light chain most frequently used in human antibodies and the pairing of that light chain in vitro with the two heavy chains of different specificity.

In another approach, a light chain could be selected by observing light chain sequences in a phage display library (for example, a phage display library comprising sequences from the variable region of the human light chain, for example, a human scFv library) and the selection of the most commonly used variable region of the light chain from among the library. Then, the light chain can be tested on the two different heavy chains of interest.

In another approach, a light chain could be selected by testing a phage display library of variable light chain sequences using the heavy chain variable sequences of both heavy chains of interest as probes. A light chain that is associated with both variable sequences of the heavy chain could be selected as a light chain for heavy chains.

In another approach, a candidate light chain could be aligned with the light chains related to the heavy chains and modifications are made to the light chain to match more closely sequence characteristics 50 common to the related light chains of both heavy chains. If it is necessary to minimize the possibilities of immunogeny, the modifications preferably result in sequences that are present in known sequences of the human light chain, so that proteolytic processing is unlikely to generate a T-cell epitope based on known parameters and methods. in the technique for the evaluation of the probability of immunogeny (i.e. computer tests, as well as wet tests). 55

All of the above approaches are based on in vitro methods that subsume a series of a priori restrictions, for example, sequence identity, ability to associate with specific preselected heavy chains, etc. There is a need in the art for compositions and methods that are not based on the manipulation of in vitro conditions, but instead employ more biologically sensible approaches to the preparation of human epitope binding proteins that include a light chain common.

In addition, therapeutic antibodies, for example, bispecific therapeutic antibodies, have some limitations in that they often require high doses to achieve the desired efficacy. This is partly due to the fact that antigen-antibody complexes are internalized in the endosome and are subject to lysosomal degradation in a process called target-mediated clearance. Therefore, there is a need in the art for methods and compositions that lead to more efficient antibody recycling, for example, bispecific antibody recycling, and prevent antibody degradation by promoting dissociation of antigen-antibody complexes in the endosomal compartment without compromising the specificity and affinity of the antibody towards the antigen.

 5

WO2011097603 describes a common light chain mouse. EP2275443 describes an antigen binding molecule capable of binding to two or more antigen molecules repeatedly. WO2011111007 describes antibodies with pH dependent antigen binding. WO09203918 and US5545806 describe transgenic non-human animals capable of producing heterologous antibodies. Chaparro-Riggers et al (JBC, 287: 11090-11097, 2012) describes the increase in serum half-life and the prolongation of cholesterol decrease in vivo by modifying the antibody with pH-sensitive binding to PCSK9. Igawa et al (Nat Biotech 28: 1203-1207, 2010) describes that the recycling of antibodies by binding to the modified pH-dependent antigen improves the duration of antigen neutralization.

Summary of the invention 15

The invention provides a genetically modified rodent comprising in its germ line an immunoglobulin light chain locus comprising (i) a single rearranged gene region of human immunoglobulin light chain variable region comprising human VL and JL segment sequences and ( ii) an immunoglobulin light chain constant region gene sequence in operative binding to the unique human immunoglobulin light chain variable region gene sequence 20,

wherein the only human region immunoglobulin light chain variable region rearranged gene sequence comprises at least one insertion or substitution performed by genetic engineering of a histidine codon that is not encoded by the corresponding VL and JL gene sequences of the strain human germinal, in which the only rearranged gene region of human immunoglobulin light chain variable region encodes an immunoglobulin chain comprising a light chain variable domain,

in which the histidine codon is in the nucleotide sequence that encodes a complementarity determining region (CDR) of the light chain variable domain,

wherein the light chain variable domain comprises at least one histidine residue at an amino acid position encoded by the at least one inserted or substituted histidine codon, and

wherein the immunoglobulin light chain encoded by the immunoglobulin light chain locus is susceptible to pairing with a plurality of immunoglobulin heavy chains selected by the rodent, in which each of the plurality of immunoglobulin heavy chains binds specifically to different epitopes when paired with the immunoglobulin light chain.

 35

In one aspect, a biological system for the generation of an antibody or a variable antibody domain that binds to a target antigen at a neutral pH is described herein, but exhibits a reduced binding of the same antigen at an acidic pH ( for example, pH 5.0-6.0). The biological system comprises a non-human animal, for example, a rodent (for example, a mouse or a rat) having a rearranged light chain sequence (for example, a rearranged V-J) comprising one or more histidine modifications. In various aspects, the one or more modifications with histidine are in the CDR3 codon of the light chain. In various aspects, the non-human animal comprises a human or humanized heavy chain immunoglobulin locus. In various aspects, the non-human animal comprises a replacement of endogenous non-human heavy chain variable gene segments with one or more human heavy chain VH, DH and JH segments in which the human segments are operatively linked to a constant region of non-human immunoglobulin. In various aspects, non-human animals with universal light chains comprising variable domains of light chain with substitutions of non-histidine residues for histidine residues are described herein. In various aspects these non-human histidine modified universal light chain animals (e.g. rodents, for example, mice) are called universal-histidine light chain mice, ULC-histidine mice (HULC mice) or HULC mice ( for its acronym in English). fifty

Thus, in one aspect, a genetically modified non-human animal is described herein which comprises in its germ line an immunoglobulin light chain locus comprising a single rearranged gene region of human immunoglobulin light chain variable region comprising human VL and JL segment sequences, in which the only rearranged sequence of the human immunoglobulin light chain variable region comprises a substitution of at least one non-histidine codon with a histidine codon. In one aspect, the only rearranged sequence of human immunoglobulin variable region is operably linked to a gene sequence of immunoglobulin light chain constant region. In one aspect, the immunoglobulin light chain constant region gene sequence is a non-human immunoglobulin light chain constant region gene sequence. In one aspect, the non-human immunoglobulin light chain constant region 60 gene sequence is an endogenous immunoglobulin light chain constant region gene sequence. In one aspect, the non-human animal lacks a variable region of functional non-rearranged immunoglobulin light chain. In one aspect, the immunoglobulin light chain locus is in an endogenous non-human immunoglobulin light chain locus.

 65

In one aspect, the animal further comprises in its germ line an immunoglobulin heavy chain locus comprising a non-rearranged immunoglobulin heavy chain variable region gene sequence comprising V segments

In one aspect, the substitution of at least one non-histidine codon with a histidine codon is in the 10 nucleotide sequence encoding a complementarity determining region (CDR). In one aspect, the replacement of at least one non-histidine codon with a histidine codon is in the nucleotide sequence encoding a CDR3. In one aspect, the substitution is one, two, three, four or more CDR3 codons. In one aspect, the only reordered sequence of human immunoglobulin light chain variable region comprised in the immunoglobulin light chain locus is derived from a human Vκ1-39 or Vκ3-20 gene segment. In one aspect, the only variable region of the rearranged human immunoglobulin light chain is derived from a Vκ1-39 / Jκ5 or Vκ3-20 / Jκ1 rearranged gene sequence. In one embodiment, the only rearranged human immunoglobulin light chain variable region is derived from a rearranged Vκ1-39 / Jκ5 gene sequence and the Vκ1-39 / Jκ5 gene sequence comprises a replacement of at least one non-histidine codon with a codon of histidine designed to express a histidine in a position selected from 105, 106, 108, 111 and a combination thereof. In another embodiment, the only rearranged human immunoglobulin light chain variable region is derived from a rearranged Vκ3-20 / Jκ1 gene sequence and the Vκ3-20 / Jκ1 gene sequence comprises a replacement of at least one non-histidine codon with a codon. of histidine designed to express a histidine in a position selected from 105, 106, 107, 109 and a combination thereof.

 25

In one aspect, the non-human animal described herein comprises a population of B cells in response to an antigen of interest that is enriched for antibodies that exhibit a decrease in dissociative half-life (t1 / 2) at an acidic pH. , in comparison to neutral pH, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times or at least about 30 times. In one aspect, the decrease in t1 / 2 at an acidic pH, compared to a neutral pH, is approximately 30 times or more.

In one aspect, the animal expresses an antibody comprising a human immunoglobulin light chain variable domain with a substitution of at least one non-histidine moiety with a histidine moiety at an amino acid position encoded by the at least one substituted codon in the gene sequence of immunoglobulin light chain variable region. In one aspect, the animal expresses an antibody that retains a substitution of at least one non-histidine moiety with a histidine moiety in an expressed human immunoglobulin light chain variable domain, despite somatic hypermutations.

 40

In one aspect, the non-human animal is a mammal. In one aspect, the mammal is a rodent, for example, a rat or a mouse. In one aspect, the non-human animal is a mouse. Thus, in one aspect, a genetically modified mouse is also described herein which comprises in its germ line an immunoglobulin light chain locus comprising a single rearranged gene region of human immunoglobulin light chain variable region comprising sequences of human VL and JL segments, in which the only rearranged sequence of the human immunoglobulin light chain variable region comprises a substitution of at least one non-histidine codon with a histidine codon. In one aspect, the mouse lacks a variable region of functional non-rearranged immunoglobulin light chain.

In one aspect, the only rearranged gene region of the human immunoglobulin 50 light chain variable region in the mouse germ line is operatively linked to an immunoglobulin light chain constant region gene sequence. In one aspect, the immunoglobulin light chain constant region gene sequence is selected from a rat or mouse immunoglobulin light chain constant region gene sequence. In one aspect, the immunoglobulin light chain constant region gene sequence is a mouse sequence. In one aspect, the immunoglobulin light chain locus is in an endogenous mouse immunoglobulin light chain locus.

In a further aspect, the mouse also comprises in its germ line an immunoglobulin heavy chain locus comprising an unordered sequence of immunoglobulin heavy chain variable region comprising VH, DH and JH segments operably linked to a region gene sequence 60 immunoglobulin heavy chain constant. In one aspect, the immunoglobulin heavy chain constant region gene sequence is a rat or mouse immunoglobulin heavy chain constant region gene sequence. In one aspect, the immunoglobulin heavy chain constant region gene sequence is a mouse sequence. In one aspect, the immunoglobulin heavy chain locus is in an endogenous mouse immunoglobulin heavy chain locus. 65

In one aspect, the mouse comprises a substitution of at least one non-histidine codon with a histidine codon in which the substitution is in the nucleotide sequence encoding a CDR. In one aspect, the substitution is in a CDR3 codon, for example, in one, two, three, four or more CDR3 codons. In one aspect, the mouse immunoglobulin light chain locus comprises the unique human immunoglobulin light chain variable region rearranged sequence derived from a human Vκ1-39 or Vκ 3-20 gene segment, for example, the only 5 rearranged sequence The immunoglobulin light chain variable region is derived from a rearranged Vκ1-39 / Jκ5 or Vκ3-20 / Jκ1 gene sequence. In one embodiment, the unique immunoglobulin light chain variable region rearranged sequence is derived from a rearranged Vκ1-39 / Jκ5 gene sequence and the Vκ1-39 / Jκ5 sequence comprises a replacement of at least one non-histidine codon with a codon of histidine designed to express a histidine in a position selected from 105, 106, 108, 111 and a combination thereof. In one embodiment, said replacement is designed to replace histidines at positions 105, 106, 108 and 111. In another embodiment, said replacement is designed to replace histidines at positions 106, 108 and 111.

In another embodiment, the unique immunoglobulin light chain variable region rearranged sequence is derived from a rearranged Vκ3-20 / Jκ1 gene sequence and the Vκ3-20 / Jκ1 sequence comprises a replacement of at least one non-histidine codon with a codon of histidine designed to express a histidine in a position selected from 105, 106, 107, 109 and a combination thereof. In one embodiment, said replacement is designed to replace histidines at positions 105, 106, 107 and 109. In another embodiment, said replacement is designed to replace histidines at positions 105, 106 and 109.

 twenty

In one aspect, the mouse described herein comprises a population of B cells in response to an antigen of interest that is enriched for antibodies that exhibit a decrease in dissociative half-life (t1 / 2) at an acidic pH, in comparison with a neutral pH, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, 25 at least about 25 times or at least about 30 times. In one aspect, the decrease in t1 / 2 at an acidic pH, compared to a neutral pH, is approximately 30 times or more.

In one aspect, the mouse described herein expresses a population of antigen-specific antibodies in response to an antigen of interest, in which all antibodies comprise (a) 30 domains of immunoglobulin light chain variables derived from the same single human light chain variable region rearranged gene sequence comprising a substitution of at least one non-histidine codon with a histidine codon and (b) immunoglobulin heavy chains comprising heavy chain variable domains derived from a segment repertoire V, D and J of human heavy chain.

 35

Also described herein is a non-human locus, for example, mouse locus, comprising a single rearranged light chain variable region gene sequence of human immunoglobulin comprising human VL and JL segment sequences, in which the only Human immunoglobulin light chain variable region rearranged gene sequence comprises a replacement of at least one non-histidine codon with a histidine codon. In one aspect, the locus is comprised in the germ line of a non-human animal. In one aspect, the locus comprises the only rearranged gene region of the human immunoglobulin light chain variable region derived from a human Vκ1-39 or Vκ3-20 gene segment, for example, derived from a Vκ1-39 / Jκ5 or Vκ3 gene sequence -20 / Jκ1 reordered. In one aspect, in which the only rearranged gene region of the human immunoglobulin light chain variable region present in the locus derives from the rearranged Vκ1-39 / Jκ5 sequence, the replacement of at least one non-histidine codon with a codon of Histidine is designed to express a histidine in a position selected from 105, 106, 108, 111 and a combination thereof. In another aspect, in which the only rearranged gene region of the human immunoglobulin light chain variable region present in the locus derives from the rearranged Vκ3-20 / Jκ1 sequence, the replacement of at least one non-histidine codon with a codon of Histidine is designed to express a histidine in a position selected from 105, 106, 107, 109 and a combination thereof. In various aspects, the non-human loci 50 described herein can be generated using methods described below to produce a genetically modified non-human animal.

In another aspect, a method for producing a non-human animal comprising a genetically modified immunoglobulin light chain locus in its germ line is described herein, in which the method comprises modifying a genome of an animal non-human to suppress or render non-functional endogenous immunoglobulin light chain segments V and J in an immunoglobulin light chain locus, and to put into the genome a single rearranged gene region of human light chain variable region comprising a substitution of at minus a non-histidine codon with a histidine codon. In one aspect, said method results in a genetically modified non-human animal comprising a population of B cells enriched for antibodies that exhibit a pH-dependent binding to the antigen of interest. In one aspect, the only reordered sequence of the human immunoglobulin light chain variable region placed in the genome derives from a human Vκ1-39 or Vκ3-20 gene sequence, for example, a Vκ1-39 / Jκ5 or Vκ3-20 gene sequence / Jκ1 reordered. Therefore, in the embodiment where the only rearranged sequence of the human immunoglobulin light chain variable region is derived from a rearranged Vκ1-65 39 / Jκ5 gene sequence, the replacement of at least one non-histidine codon with a histidine codon It is designed to express a histidine in a position selected from 105, 106, 108, 111 and a combination thereof. In one embodiment in which the unique human immunoglobulin light chain variable region rearranged sequence is derived from a rearranged Vκ3-20 / Jκ1 gene sequence, the replacement of at least one non-histidine codon with a histidine codon is designed to express a histidine in the one position selected from 105, 106, 107, 109 and a combination thereof. 5

In another aspect, a method of generating an antibody that exhibits pH-dependent binding to an antigen of interest is described herein which comprises (a) generating a mouse that is described herein (eg, a mouse that it comprises in its germ line an immunoglobulin light chain locus comprising a single rearranged light chain variable region sequence of human immunoglobulin comprising human VL and JL segment sequences and a replacement of at least one non-histidine codon with a histidine codon in its light chain variable region rearranged sequence), (b) immunize the mouse with an antigen of interest and (c) select an antibody that binds to the antigen of interest with a desired affinity at a neutral pH showing the at the same time a reduced binding to the antigen at an acidic pH. In one aspect, the method results in a generation of an antibody that has a t1 / 2 at acidic pH and 15 37 ° C of about 2 minutes or less. In one aspect, the method results in a generation of an antibody that shows a decrease in dissociative half-life (t1 / 2) at an acidic pH, compared to a neutral pH, of at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times or at least about 30 20 times.

In other aspects, additional methods of generating an antibody having pH dependent binding to an antigen of interest are described herein. Such a method comprises (a) selecting a first antibody that binds to an antigen of interest with a desired affinity, (b) modifying an immunoglobulin light chain 25 nucleotide sequence of the first antibody to comprise a substitution of minus a non-histidine codon with a histidine codon, (c) expressing an immunoglobulin heavy chain of the first antibody and the modified immunoglobulin light chain in a cell and (d) selecting a second antibody expressed in the cell that retains an affinity desired by the antigen of interest at neutral pH and show a reduced binding to the antigen of interest at an acidic pH. In one aspect, the immunoglobulin light chain nucleotide sequence of the first antibody comprises a unique rearrangement of human immunoglobulin light chain variable region. In one aspect, the first antibody is generated in a non-human animal, for example, a mouse, comprising an immunoglobulin light chain sequence derived from a single rearranged human immunoglobulin light chain variable region sequence and the modification of the light chain of immunoglobulin in the only rearranged sequence of human immunoglobulin 35 variable region. In one aspect, the first antibody is generated in a non-human animal, for example, a mouse, which additionally comprises an immunoglobulin heavy chain sequence derived from a repertoire of human VH, DH and JH segments. In one aspect, the only rearranged sequence of the human immunoglobulin light chain variable region is selected from the gene sequence Vκ1-39 / Jκ5 and Vκ3-20 / Jκ1. In one embodiment, in which the only rearranged sequence of the human immunoglobulin light chain variable region is Vκ1-40 39 / Jκ5, the modification in the immunoglobulin light chain nucleotide sequence of the first antibody is made in the CDR3 codon in a position selected from 105, 106, 108, 111 and a combination thereof. In one embodiment in the single rearrangement sequence of the human immunoglobulin light chain variable region is Vκ3-20 / Jκ1, the modification in the immunoglobulin light chain nucleotide sequence of the first antibody is made in the CDR3 codon at a position selected from 105, 106, 107, 109 and a combination thereof.

In one aspect, the method of generating an antibody that exhibits pH-dependent binding to an antigen of interest described herein results in an antibody that shows a decrease in dissociative half-life (t1 / 2) at a pH. acid, compared to a neutral pH, of at least about 2 50 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times or at least about 30 times. In one aspect, the method of generating the antibody results in an antibody that has a t1 / 2 at acidic pH and 37 ° C of about 2 minutes or less. 55

Any of the embodiments and aspects described herein may be used in combination with each other, unless otherwise indicated or evident from the context. Other embodiments will be apparent to those skilled in the art from a review of the following description.

 60

Brief description of the drawings

FIG. 1 illustrates an amino acid alignment of light chains derived from human Vκ1-39 of various antigen-specific antibodies (A-K antibodies). The histidine (H) residues located within each light chain sequence are in bold. Various light chain regions (conserved framework region and 65 CDR) are indicated above the alignment.

FIG. 2 illustrates the combinations and locations of histidine residues introduced by genetic engineering in the CDR3 region of light chains derived from human Vκ1-39 by mutagenesis. The corresponding nucleic acid sequences are included. Histidine residues introduced through mutagenesis and corresponding nucleic acid residues are shown in bold. The positions of the amino acids (105, 106, etc.) are based on a singular numbering described in Lefranc et al. (2003) Dev. Comp. 5 Immunol. 27: 55-77 and can also be seen at www.imgt.org.

FIG. 3 illustrates the level of antibody expression in ng / ml detected in supernatants of CHO cells transfected with nucleic acids encoding five (1-5) different heavy chains and light chains derived from Vκ1-39 that had engineering-introduced histidine residues genetics in the 10 places indicated (see Y axis) in CDR3.

FIG. 4 is a western blot showing the expression of selected antigen specific human antibodies containing light chains modified by genetic engineering with histidine in CHO cell supernatants. fifteen

FIG. 5A-5E show the binding kinetics for selected heavy chains (1-5) of antigen specific antibodies paired with various light chains modified by genetic engineering with histidine at a neutral pH (7.4) and one (5.75). Various kinetic parameters are shown including kA, Kd, KD and t1 / 2. SU = no union. twenty

FIG. 6 shows kinetic parameters (KD and t1 / 2) for antibodies comprising parental universal light chain or universal light chain modified by genetic engineering with histidine paired with indicated heavy chains (2, 3 and 6). Histidine substitutions lead to a strong dependence on pH in several antibodies. Histidine substitutions were made in CDR3 to convert the sequence 105QQSYSTP111 (SEQ ID NO: 3) to 105HHSYSTH111 (SEQ ID NO: 5). Note that SU = no junction detected (KD> 10 micromolar).

FIG. 7 shows the sequence and properties (% of GC content, N,% of disappearance, Tm) of selected mutagenesis primers used for genetically engineered histidine residues in CDR3 of a rearranged light chain sequence Vκ1-39 / Jκ5 human. SEQ ID NOs are included for these primers used in the Sequence Listing included in the Table below. F = direct primer, R = reverse primer.

FIG. 8A-8B show a general strategy for the construction of direction vectors for the genetic introduction of histidine residues into a rearranged sequence of human light chain variable region derived from Vκ1-39 / Jκ5 variable region to make a mouse genetically modified that expresses antibodies that contain the modified human light chain. FIG. 8C-8D show the introduction of the direction vector for ULC-H105 / 106/108/111 substitutions in ES cells and the generation of heterozygous mice therefrom; while FIG. 8E-8F show the introduction of the direction vector for ULC-H106 / 108/111 substitutions in ES cells and the generation of heterozygous mice therefrom. Diagrams are not presented to scale. Unless otherwise indicated, filled forms and solid lines represent a mouse sequence, empty forms and double lines represent a human sequence.

 Four. Five

FIG. 9 shows antiserum titers against immunogen of heterozygous mice for the universal histidine light chain (HULC) (with 4 substitutions of His-mice HULC 1927; with 3 substitutions of His-mice HULC 1930) and wild-type animals in a second blood draw

FIG. 10 is a comparison of the number of clones positive for total antigen and the number of clones 50 positive for antigen presenting binding to pH-sensitive antigen, obtained from heterozygous HULC hybridoma fusions (1927 vs. 1930) and TS mice. The figure includes data for two mice for each type of mouse ("mouse 1" and "mouse 2").

FIG. 11A-11C show sensograms of surface plasmon resonance binding experiments in which monoclonal antibodies (AA, BB, CC, DD, HH, GG, NN and OO) of heterozygous TS or HULC mice were allowed to associate with the immunogen at neutral pH (pH 7.4), followed by a change to a buffer with pH 7.4 or 6.0 for the dissociation phase. The individual lines in each graph represent the binding responses at different concentrations of the respective antibodies. All experiments were performed at 25 ° C. The values of dissociative half-life (t1 / 2) are indicated above the respective sensograms and the change in number 60 times (t1 / 2) is included to the right of each sensogram. The AA, BB, CC, DD, HH and GG antibodies were from 1927 heterozygous HULC mice using His-substituted light chain, NN is from 1927 heterozygous HULC mouse using TS light chain and OO is from a TS mouse (See Table 4 for clarifications).

FIG. 12 shows positions of histidine residues introduced by genetic engineering in the 65 CDR3 region of light chains derived from Vκ3-20 by mutagenesis. Histidine residues introduced through mutagenesis and corresponding nucleic acid residues are shown in bold. The amino acid positions (105, 106, etc.) are based on a unique numbering described in Lefranc et al. (2003) Dev. Comp. Immunol 27: 55-77 and can also be seen at www.imgt.org.

FIG. 13 shows the sequence and properties (% of GC content, N,% of disappearance, Tm) of 5 selected mutagenesis primers used for genetically engineered histidine residues in CDR3 of a rearranged light chain sequence Vκ3-20 / Jκ1 human. SEQ ID NOs are included for these primers used in the Sequence Listing included in the Table below. D = direct primer, I = reverse primer.

 10

FIG. 14A-14B show a general strategy for the construction of direction vectors for the genetic introduction of histidine residues in a rearranged sequence of human light chain variable region derived from Vκ3-20 / Jκ1 variable region to make a modified mouse genetically expressing antibodies that contain the modified human light chain. FIG. 14C shows the introduction of the direction vector for ULC-Q105H / Q106H / Y107H / S109H substitutions in 15 ES cells and the generation of heterozygous mice therefrom; while FIG. 14D shows the introduction of the direction vector for ULC-Q105H / Q106H / S109H substitutions in ES cells and the generation of heterozygous mice therefrom. Diagrams are not presented to scale. Unless otherwise indicated, filled forms and solid lines represent a mouse sequence, empty forms and double lines represent a human sequence. twenty

Detailed description of the invention

Definitions

 25

This document describes non-human animals (for example, mice, rats, rabbits, hamsters, etc.) genetically modified that comprise in their genome, for example, in their germ line, a nucleotide sequence or sequences encoding molecules of human antibodies that exhibit pH-dependent antigen binding, for example, an immunoglobulin light chain nucleotide sequence comprising a rearranged human immunoglobulin light chain variable region sequence encoding antibodies that have pH-dependent antigen binding; embryos, cells and tissues that comprise them; manufacturing methods thereof; as well as methods of using them. Unless otherwise defined, all terms and phrases used herein include the meanings that the terms and phrases have reached in the technique, unless clearly stated otherwise or clearly evident from the context in which The term or phrase is used. 35

The term "antibody", as used herein, includes immunoglobulin molecules comprising four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable domain and a heavy chain constant region (CH). The constant region of the heavy chain comprises three domains, CH1, CH2 and CH3. Each light chain comprises a variable light chain domain and a constant light chain region (CL). The variable domains of the heavy chain and the light chain can be further subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with regions that are more conserved, called conserved framework regions (FR). . Each heavy chain and light chain variable domain consists of three CDRs and four FRs, arranged from the amino terminal end to the carboxy terminal end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (CDR Heavy chain can be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDR can be abbreviated as LCDR1, LCDR2 and LCDR3). The term "high affinity" antibody refers to an antibody that has a KD with respect to its target epitope of about 10-9 M or less (for example, about 1 x 10-9 M, 1 x 10-10 M, 1 x 10-11 M or 50 approximately 1 x 10-12 M). In one embodiment, KD is measured by surface plasmon resonance, for example, BIACORE ™; In another embodiment, KD is measured by ELISA.

The phrase "bispecific antibody" includes an antibody capable of selectively binding two or more epitopes. Bispecific antibodies generally comprise two non-identical heavy chains, each heavy chain specifically binding to a different epitope - either in two different molecules (e.g., different epitopes in two different immunogens) or in the same molecule (e.g., different epitopes in the same immunogen). If a bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one or two or three or four or more orders of magnitude less than the affinity of the first heavy chain by 60 the second epitope and vice versa. Epitopes specifically bound by the bispecific antibody can be on the same or a different target (for example, on the same or a different protein). Exemplary bispecific antibodies include those with a first heavy chain specific for a tumor antigen and a second heavy chain specific for a cytotoxic marker, for example, an Fc receptor (e.g., FcγRI, FcγRII, FcγRIII, etc.) or a marker of T lymphocytes (for example, CD3, CD28, etc.). Additionally, the second heavy chain variable domain may be substituted with a heavy chain variable domain having a different desired specificity. For example, a bispecific antibody with a first heavy chain specific for a tumor antigen and a second heavy chain specific for a toxin can be paired in order to deliver a toxin (eg, saporin, vinca alkaloid, etc.) to a tumor cell Other exemplary bispecific antibodies include those with a first heavy chain specific for an activating receptor (eg, B cell receptor, FcγRI, FcγRIIA, FcγRIIIA, FcαRI, T lymphocyte receptor, etc.) and a second heavy chain specific for an inhibitor receptor (for example, FcγRIIB, CD5, CD22, CD72, CD300a, etc.). Such bispecific antibodies can be constructed for therapeutic conditions associated with cell activation (eg, allergy and asthma). Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same immunogen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same immunogen can be fused to nucleic acid sequences encoding the same or different heavy chain constant regions, and said sequences can be expressed in a cell that expresses a light chain of immunoglobulin. A typical bispecific antibody has two heavy chains each having three heavy chain CDRs, followed by (from the N-terminus to the C-terminus) a C domain

 twenty

The term "cell" includes any cell that is suitable for expressing a recombinant nucleic acid sequence. Cells include those of prokaryotes and eukaryotes (single cell or multi-cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., Etc.), mycobacterial cells, fungal cells, yeast cells (e.g. S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, insect cells infected with baculovirus, Trichoplusia 25 ni, etc.), non-human animal cells, human cells or cell fusions such as, for example, hybridomas or quadromas. In some embodiments, the cell is a human, monkey, ape, hamster, rat or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the following cells: CHO (for example, CHO K1, DXB-11 CHO, Veggie-CHO), COS (for example, COS-7), retinal cells, Vero, CV1 , kidney (for example, HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, 30 HL-60, (for example, BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, 3A BRL cells, HT1080 cells, myeloma cells, tumor cells and a cell line derived from a cell mentioned above. In some embodiments, the cell comprises one or more viral genes, for example, a retinal cell that expresses a viral gene (for example, a PER.C6 ™ cell). 35

The phrase "complementarity determining region," or the term "CDR," includes an amino acid sequence encoded by an immunoglobulin gene nucleic acid sequence of an organism that normally (i.e., in a wild-type animal) appears between two framework regions conserved in a variable region of a light or a heavy chain of an immunoglobulin molecule (for example, an antibody or a T-lymphocyte receptor). A CDR may be encoded, for example, by a germ line sequence or a rearranged or non-rearranged sequence, and, for example, by a B cell or a virgin or mature T lymphocyte. A CDR can be somatically mutated (for example, it can vary from a sequence encoded in the germ line of an animal), humanized and / or modified with amino acid substitutions, additions or deletions. In some circumstances (for example, for a CDR3), the CDRs may be encoded by two or more sequences (for example, germ line sequences) that are not contiguous (for example, in a non-rearranged nucleic acid sequence) but which are contiguous in a B cell nucleic acid sequence, for example, as a result of splicing or connecting the sequences (eg, VDJ recombination to form a CDR3 heavy chain).

 fifty

The term "conservative," when used to describe a conservative amino acid substitution, includes the substitution of an amino acid residue for another amino acid residue that has a side chain R group with similar chemical properties (eg, loading or hydrophobia). In general, a conservative amino acid substitution will not substantially change the functional properties of interest of a protein, for example, the ability of a variable region to specifically bind to a target epitope with a desired affinity. Examples of amino acid groups having side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic hydroxyl side chains such as serine and threonine; side chains containing amide such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine and tryptophan; basic side chains such as lysine, arginine and histidine; acidic side chains such as aspartic acid and glutamic acid; and the 60 sulfur-containing side chains such as cysteine and methionine. Conservative amino acid substitution groups include, for example, valine / leucine / isoleucine, phenylalanine / tyrosine, lysine / arginine, alanine / valine, glutamate / aspartate and asparagine / glutamine. In some embodiments, a conservative amino acid substitution may be the substitution of any native moiety in a protein with alanine, as used, for example, in alanine tracking mutagenesis. In some embodiments, a conservative substitution 65 is made that has a positive value in the log matrix of PAM250-probability disclosed in Gonnet et al. (1992), Science 256: 1443-1445. In some embodiments, the substitution is a moderately conservative substitution in which the substitution has a non-negative value in the PAM250-probability log matrix.

In some embodiments, the positions of residues in a light chain or an immunoglobulin heavy chain differ in one or more conservative amino acid substitutions. In some embodiments, the positions of 5 residues in an immunoglobulin light chain or functional fragment thereof (for example, a fragment that allows expression and secretion from, for example, a B cell) are not identical to those of a chain lightweight whose amino acid sequence is listed herein, but differs in one or more conservative amino acid substitutions.

 10

The phrase "epitope binding protein" includes a protein that has at least one CDR and that is capable of selectively recognizing an epitope, for example, is capable of binding to an epitope with a KD that is approximately one micromolar or less ( for example, a KD that is about 1 x 10-6 M, 1 x 10-7 M, 1 x 10-9 M, 1 x 10-9 M, 1 x 10-10 M, 1 x 10-11 M or about 1 x 10-12 M). Therapeutic epitope binding proteins (for example, therapeutic antibodies) often require a KD that is in the nanomolar or picomolar range.

The phrase "functional fragment" includes epitope-binding protein fragments that can be specifically expressed, secreted and bound to an epitope with a KD in the micromolar, nanomolar or picomolar range. Specific recognition includes having a KD that is at least in the micromolar range, the nanomolar range 20 or the picomolar range.

The term "germ line" in reference to an immunoglobulin nucleic acid sequence includes a nucleic acid sequence that can be passed to the progeny.

 25

The phrase "heavy chain" or "immunoglobulin heavy chain" includes an immunoglobulin heavy chain sequence, including the immunoglobulin heavy chain constant region sequence, of any organism. Variable heavy chain domains include three heavy chain CDRs and four FR regions, unless otherwise specified. Heavy chain fragments include CDR, CDR and FR and combinations thereof. A typical heavy chain has, after the variable domain (end N to 30 end C), a CH1 domain, a hinge, a CH2 domain and a CH3 domain. A functional fragment of a heavy chain includes a fragment that is capable of specifically recognizing an epitope (for example, recognizing the epitope with a KD in the micromolar, nanomolar or picomolar range), which is capable of expressing and secreting from a cell and which Understand at least one CDR. A heavy chain variable domain is encoded by a variable region gene sequence, which generally comprises VH, DH and JH segments derived from a repertoire of VH, DH and JH segments present in the germ line. Sequences, locations and nomenclature for heavy chain segments V, D and J for various organisms can be found in the IMGT database, www.imgt.org.

The term "identity", when used in relation to a sequence, includes identity as determined by a series of different algorithms known in the art that can be used to measure nucleotide and / or amino acid sequence identity. In some embodiments described herein, identities are determined using a ClustalW v alignment. 1.83 (slow) using an open gap penalty of 10.0, a gap extension penalty of 0.1, and using a Gonnet similarity matrix (MACVECTOR ™ 10.0.2, MacVector Inc., 2008). The length of the sequences compared with respect to the identity of the sequences will depend on the particular sequences, but in the case of a constant light chain domain, the length should contain a sequence of sufficient length to fold into a constant chain domain. light that is capable of self-association to form a constant domain of canonical light chain, for example, capable of forming two beta sheets comprising beta chains and capable of interacting with at least one CH1 domain of a human being or a mouse. In the case of a CH1 domain, the length of the sequence should contain a sequence of sufficient length to fold into a CH1 domain that is capable of forming two beta sheets comprising beta chains and capable of interacting with at least one constant domain of light chain of a mouse or a human being.

The phrase "immunoglobulin molecule" includes two heavy chains of immunoglobulin and two light chains of immunoglobulin. Heavy chains may be identical or different and light chains may be identical or different.

The "light chain" phrase includes an immunoglobulin light chain sequence of any organism and, unless otherwise specified, includes the human kappa and lambda light chains and a VpreB, as well as 60 substitute light chains. Variable light chain domains typically include three light chain CDRs and four conserved framework regions (FR), unless otherwise specified. In general, a full length light chain includes, from the amino end to the carboxyl end, a variable domain that includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and a constant light chain region. A light chain variable domain is encoded by a light chain variable region gene sequence, which comprises, in general, 65 segments VL and JL, derived from a repertoire of segments V and J present in the germ line. Sequences, locations and nomenclature for light chain segments V and J for various organisms can be found in the IMGT database, www.imgt.org. Light chains include those, for example, that do not selectively bind either to a first or second epitope selectively linked by the epitope binding protein in which they appear. Light chains also include those that bind and recognize, or help the heavy chain with binding and recognition of, one or more epitopes selectively linked by the epitope binding protein in which they appear. Common or universal light chains include those derived from a human Vκ1-39Jκ5 gene or a human Vκ3-20Jκ1 gene, and include somatically mutated (eg, affinity matured) versions thereof.

The phrase "micromolar interval" is intended to mean 1-999 micromolar; the phrase "nanomolar interval" is intended to mean 1-999 nanomolar; The phrase "picomolar interval" is intended to mean 1-999 picomolar.

The phrase "somatically mutated" includes the reference to a nucleic acid sequence of a B cell that has undergone class change, in which the nucleic acid sequence of an immunoglobulin variable region (eg, nucleotide sequence encoding a heavy chain variable domain or that includes a heavy chain CDR or FR sequence) in cell B changed class is not identical to the nucleic acid sequence in cell B before class change, such as, for example, a difference in a CDR nucleic acid sequence or conserved framework region between a B cell that has not undergone class change and a B cell that has undergone class change. "Somatically mutated" includes the reference to affinity matured B-cell nucleic acid sequences that are not identical to the corresponding immunoglobulin variable region sequences in B cells that are not affinity-matured (ie, sequences in the genome of germ line cells). The phrase "somatically mutated" also includes the reference to an immunoglobulin variable region nucleic acid sequence of a B cell after exposure of the B cell to an epitope of interest, in which the nucleic acid sequence differs from the corresponding nucleic acid sequence before exposure of cell B to the epitope of interest. The phrase "somatically mutated" refers to antibody sequences that have been generated in an animal, for example, a mouse having nucleic acid sequences of human immunoglobulin variable region, in response to an immunogenic exposure, and which are a result. of the operative selection processes inherently in an animal of this type.

The term "non-rearranged", with reference to a nucleic acid sequence, includes nucleic acid sequences that exist in the germ line of an animal cell.

The phrase "variable domain" includes an amino acid sequence of an immunoglobulin light or heavy chain (modified as desired) comprising the following amino acid regions, in sequence from end N to end C (unless otherwise indicated) : FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. 35

The term "operably linked" refers to a relationship in which the operably linked components function in the intended manner. In one case, a nucleic acid sequence encoding a protein may be operably linked to regulatory sequences (eg, promoter, enhancer, silencer sequence, etc.) in order to preserve the appropriate transcriptional regulation. In one case, a nucleic acid sequence of an immunoglobulin variable region (or segments V (D) J) may be operably linked to a nucleic acid sequence of an immunoglobulin constant region in order to allow appropriate recombination between the sequences in an immunoglobulin heavy or light chain sequence.

The term "replacement" in reference to gene replacement refers to the placement of exogenous genetic material in an endogenous genetic locus, thereby replacing all or a portion of the endogenous gene with an orthologous or homologous nucleic acid sequence.

"Functional" as used herein, for example, in reference to a functional polypeptide, includes a polypeptide that retains at least one biological activity normally associated with the native protein. In another case, a functional immunoglobulin gene segment may include a variable gene segment that is susceptible to productive rearrangement to generate a rearranged immunoglobulin gene sequence.

"Neutral PH" includes a pH between about 7.0 and about 8.0, for example, a pH between about 7.0 and about 7.4, for example, between about 7.2 and about 7.4, for example, the physiological pH. "Acid PH" includes a pH of 6.0 or less, for example, a pH between about 5.0 and about 6.0, a pH between about 5.75 and about 6.0, for example, a pH of endosomal or lysosomal compartments.

Histidine residues modified by genetic engineering in immunoglobulin chain genes 60

The inventors have discovered that non-human animals can be made that express antibodies that are capable of binding to an antigen in a pH-dependent manner by making modifications of a variable region of immunoglobulin light chain at one or more positions along the sequence of the light chain Methods for making modifications in the germ line of a non-human animal are described so that the animal expresses histidines in antibody CDR. In particular, methods are described for making modifications in a variable sequence of immunoglobulin light chain in the mouse germ line. The sequence of the variable region, for example, of light chains, typically shows somatic hypermutation along the sequence of the variable region and, in some cases, such mutations can result in a replacement of histidine residues (see, for example, FIG. 1). Such mutations can occur even in the complementarity determining regions (CDR), which are the regions of the variable domains responsible for antigen binding. In some cases, such mutations may result in antibodies that show a pH-dependent antigen binding, for example, a reduced antigen binding at an acidic pH compared to antigen binding at a neutral pH. Such pH-dependent antigen binding is desired since it can allow the antibody to bind to the antigen outside the cell, and, when internalized in an endosome, release the antigen and recycle back to the surface to bind to another antigen. , avoiding the clearance mediated by the target. Approaches to the introduction of histidine residues to achieve this effect have been published by using a random histogenesis to genetically engineer pH-dependent binding properties in anti-IL-6R antibodies (US 2011 / 0111406 A1). However, random mutagenesis of antibody residues can result in reduced affinity of the antibody to the antigen. A genetically modified non-human animal for the expression of a histidine substitution in the sequence of the antibody allows the generation of high affinity antibodies in response to an antigen of interest that, due to the modification or modifications with histidine, would also show a binding a pH dependent antigen.

Therefore, in various aspects of this document a genetically modified non-human animal is described (for example, a rodent, for example, a mouse or a rat) comprising in its genome, for example, in its germ line, a human immunoglobulin light chain variable region sequence comprising modifications that result in the animal expressing antibodies capable of binding antigens in a pH dependent manner. In one aspect, the non-human animal comprises modifications in the human immunoglobulin light chain variable region sequence (eg, VL and / or JL segment sequence) comprising substitutions in at least one non-histidine codon with a codon of histidine (in some 25 cases, they may also be referred to as "histidine substitution", "histidine codon substitution" or the like). In one aspect, the animal comprises at least one substitution of a non-histidine codon with a histidine codon in a nucleotide sequence of a complementarity determining region (CDR, for example, CDR1, CDR2 and / or CDR3) of a light chain of human immunoglobulin. In one aspect, the substitution is in a CDR3 codon. In one aspect, the light chain is a κ light chain. In one aspect, the animal expresses an immunoglobulin light chain, for example, a light chain CDR, for example, a light chain CDR3, comprising a substitution of at least one amino acid with a histidine. In another aspect, the light chain is a light chain λ. In yet another aspect, the mouse comprises a substitution of at least one non-histidine codon with a histidine codon in both κ and λ light chains.

 35

The histidine residue is encoded by two different codons, CAT and CAC (deoxyribonucleic acid residues). Therefore, a non-histidine codon can be substituted with a CAT or a CAC. The substitution is engineered in a codon that in its germ line configuration (i.e., non-somatically mutated state) does not encode a histidine residue.

 40

In one aspect a light chain is a universal light chain (also called a common light chain). As described in U.S. Patent Applications No. 13 / 022,759, 13 / 093,156, 13 / 412,936 and 13 / 488,628 (U.S. Application Publications No. 2011/0195454, 2012/0021409, 2012/0192300 and 2013/0045492), an animal Non-human (for example, a mouse) that selects a common light chain for a plurality of heavy chains has practical utility. In various aspects, antibodies expressed in a non-human animal comprising only one common light chain will have heavy chains that can be associated and expressed with an identical or substantially identical light chain. This is particularly useful in the preparation of bispecific antibodies. For example, such an animal can be immunized with a first immunogen to generate a B cell that expresses an antibody that specifically binds to a first epitope. The animal (or a genetically equal animal) can be immunized with a second immunogen to generate a B cell that expresses an antibody that specifically binds to the second epitope. Variable heavy chain regions can be cloned from B cells and expressed with the same heavy chain constant region and the same light chain (eg, a common light chain) in a cell to produce a bispecific antibody, in which the heavy chain component of the bispecific antibody has been selected by an animal to be associated and expressed with the same light chain component. In various aspects described, the variable regions of genetically modified mice are human variable regions.

Therefore, a mouse that is capable of generating immunoglobulin light chains that will conveniently pair with a very diverse family of heavy chains, including heavy chains whose human variable regions deviate from the germ line sequences, was modified genetically, 60 by For example, variable regions matured by affinity or somatically mutated. In various aspects, the mouse was intended to match human light chain variable domains with human heavy chain variable domains comprising somatic mutations, thereby allowing a pathway to high affinity binding proteins suitable for use as therapeutic agents of use. human.

 65

The genetically engineered mouse, through the long and complex process of antibody selection within an organism, makes biologically appropriate choices in pairing a diverse collection of human heavy chain variable domains with a limited number of light chain options human. In order to achieve this, the mouse is genetically engineered to present a limited number of human light chain variable domain options along with a wide variety of human heavy chain variable domain options. Following exposure with an immunogen, the mouse maximizes the number of solutions in its repertoire to develop an antibody to the immunogen, limited largely or solely by the number or light chain options in its repertoire. In various aspects, this includes allowing the mouse to achieve adequate and compatible somatic mutations of the light chain variable domain which, however, will be compatible with a relatively large diversity of human heavy chain variable domains, including, in particular, variable domains heavy chain human somatically mutated.

The common light chain mouse modified by genetic engineering described in US Application Publications No. 2011/0195454, 2012/0021409, 2012/0192300 and 2013/0045492 comprised a nucleic acid sequence encoding a limited repertoire of light chain options, for example, a common or universal "ULC" light chain. unviersal light chain "comprising no more than two VL segments or a single reordered sequence of human immunoglobulin light chain variable region. To achieve said limited repertoire, a mouse was genetically engineered to render its capacity non-functional or substantially non-functional to make, or reorganize, a native mouse light chain variable domain. In one aspect, this was achieved, for example, by suppressing the mouse light chain variable region gene segments. As described above, the endogenous mouse locus can then be modified by suitable exogenous human light chain variable region gene segments of choice n, operatively linked to the endogenous mouse light chain constant domain, so that exogenous human variable region gene segments can combine with the endogenous mouse light chain constant region gene and form a rearranged reverse chimeric light chain gene (human variable, mouse constant). In 25 different aspects, the variable light chain region is capable of mutating somatically. In various aspects, to maximize the ability of the light chain variable region to acquire somatic mutations, the appropriate enhancer or enhancers are retained in the mouse. In one aspect, in the modification of a mouse κ light chain locus to replace mouse κ light chain gene segments with human κ light chain gene segments, the mouse κ intronic enhancer and the 3 κ mouse enhancer are they keep 30 functionally or uninterrupted.

Therefore, a genetically modified mouse that expresses a limited repertoire of reverse chimeric light chains (human variable, mouse constant) associated with a variety of chimeric heavy chains (human variable, mouse constant) inverse was described. In various aspects, the endogenous mouse κ light chain 35 gene segments are suppressed and replaced with a single (or two) rearranged human light chain region, operably linked to the endogenous mouse Cκ gene. In aspects, to maximize the somatic hypermutation of the rearranged human light chain region, the mouse κ intronic enhancer and 3 'κ mouse enhancer are maintained. In various aspects, the mouse also comprises a non-functional light chain locus A or a deletion thereof or a deletion that makes the locus unable to make a light chain A. 40

The antibodies generated in a universal light chain mouse in response to various antigens that were able to use a diverse repertoire of heavy chain variable region sequences, comprising a diverse repertoire of VH, DH and JH segments. The antibodies generated in said genetically engineered ULC mouse are useful for designing bispecific therapeutic antibodies; however, as with any other antibody, each bispecific antibody can only bind to one target during its lifetime in plasma; The antibody is internalized in an endosome and targets lysosomal degradation. Studies have shown that the FcRn receptor type MHC class I FcRn is capable of rescuing immunoglobulins from lysosomal degradation by returning it to the cell surface from the classification endosome. Simister and Mostov (1989) An Fc receptor structurally related to MHC class I antigens. Nature 337: 184-87. As explained above, in order to improve the efficiency of antibody recycling, additional modifications of the antibody sequences are beneficial, for example, modifications that result in decreased antigen binding at acidic pH (eg, pH of the endosome), while retaining affinity and antibody-antigen specificity at neutral pH (for example, physiological pH). The non-human animals described herein, in which non-histidine residues are replaced by histidine residues in the sequence of a universal light chain, are beneficial because they are capable of producing high affinity antibodies based on the format Universal light chain that also show pH dependent binding, for example, show reduced binding to the antigen at an acidic pH versus neutral pH.

Therefore, in one aspect, this document describes a non-human animal (for example, a rodent, for example, a mouse or a rat) comprising in its genome, for example, in its germ line, a repertoire limited of human light chain variable regions or a single human light chain variable region, of a limited repertoire of human light chain variable gene segments, in which the human light chain variable region or the human light chain variable regions comprise at least one substitution of a non-histidine codon with a histidine codon. In some aspects, provided that non-human animals are genetically modified to include a single non-rearranged gene segment of human light chain variable region (or two human light chain variable region gene segments) that is rearranged to form a human light chain variable region rearranged gene (or two light chain variable region rearranged genes) expressing a single light chain (or expressing a two of two light chains), in which the variable region gene or genes of Light chain comprises a substitution of at least one non-histidine codon with a histidine codon. The human light chain rearranged variable domains encoded by this histidine-substituted light chain variable region gene or genes are capable of pairing with a plurality of affinity-matured human heavy chains selected by the animals, in which the variable regions of Heavy chain specifically bind to different epitopes. In various aspects, the at least one substitution of a non-histidine moiety with a histidine moiety results in a rearranged human light chain that, when expressed with a related heavy chain, binds to its antigen in a manner dependent on the pH

Genetically modified animals are described that express a limited repertoire of human light chain variable domains or a single human light chain variable domain, of a limited repertoire of human light chain variable region gene sequences, in which the region gene sequences Variables comprise at least one substitution of a non-histidine codon with a histidine codon. In some aspects, the described animals are genetically modified to include a single human V / J light chain sequence (or two V / J sequences) comprising a substitution of at least one non-histidine codon with a histidine codon and expresses a variable region of a single light chain (or expressing one or both of two variable regions). In one aspect, a light chain comprising the variable sequence is capable of pairing with a plurality of affinity-matured human heavy chains selected clonally by the animal, in which the heavy chain variable regions specifically bind different epitopes. In one aspect, the antibody binds to its antigen or antigens in a pH dependent manner. In one aspect, the only human V / J light chain sequence is selected from Vκ1-39Jκ5 and Vκ3-20Jκ1. In one aspect, the two V / J sequences are Vκ1-39Jκ5 and Vκ3-20Jκ1. In one aspect, the Vκ1-39Jκ5 and Vκ3-20Jκ1 sequences are rearranged 25 V / J sequences.

In one aspect, a genetically modified non-human animal comprising a single human immunoglobulin light chain VL gene segment that is capable of rearranging with a human JL gene segment (selected from one or a plurality of JL segments) is described herein. ) and that encodes a human variable domain of an immunoglobulin light chain, in which the single human immunoglobulin light chain VL gene segment and / or human JL gene segment comprise a substitution of at least one non-histidine codon with a histidine codon. In another aspect, a genetically modified mouse comprising no more than two human VL gene segments is described herein, each of which is capable of rearranging with a human JL gene segment (selected from one or a plurality of 35 segments JL) and encoding a human variable domain of an immunoglobulin light chain, in which each of the no more than two VL gene segments and / or the JL gene segment comprise a substitution of at least one non-histidine moiety with a histidine residue

This document also describes a genetically modified non-human animal that comprises in its genome, for example, in its germ line, a unique rearranged sequence of the human immunoglobulin light chain variable region comprising human VL and JL sequences in the genome. that the only rearranged human immunoglobulin light chain variable region comprises a replacement of at least one non-histidine codon with a histidine codon. In one aspect, the only rearranged sequence of the human immunoglobulin light chain variable region is derived from VL and JL gene sequences of human germ line, except for histidine substitution or substitutions. In one aspect, the human immunoglobulin light chain is a human immunoglobulin κ chain. Thus, in one aspect, the human VL gene sequence is selected from Vκ1-39 and Vκ3-20. In one aspect, the only human immunoglobulin light chain variable region rearranged sequence comprises a Vκ1-39 / J or Vκ3-20 / J rearranged sequence. In one aspect, the human JL gene sequence is selected from Jκ1, Jκ2, Jκ3, Jκ4 and Jκ5. In one aspect the human JL sequence is selected from Jκ1 and Jκ5. In one aspect, the only rearranged sequence of the human immunoglobulin light chain variable region is selected from Vκ1-39Jκ5 and Vκ3-20Jκ1 (for example, except for substitution or substitutions with histidine). In an alternative aspect, the human immunoglobulin light chain is a human λ chain.

In one aspect, the substitution of at least one non-histidine codon with a histidine codon is in the nucleotide sequence that encodes a complementarity determining region (CDR) of the light chain variable domain. In one aspect, the replacement of at least one non-histidine codon with a histidine codon is in the nucleotide sequence encoding CDR1, CDR2 or CDR3 of the light chain variable domain. In a specific aspect, the substitution is in the nucleotide sequence encoding CDR3.

 60

In one aspect, the substitution is at least one non-histidine codon with a histidine codon in the CDR3 codon of the gene sequence of the variable region of the human light chain. In one embodiment, the substitution is one, two, three, four or more CDR3 codons. In the embodiment in which the only rearranged human immunoglobulin light chain variable region is a Vκ1-39Jκ5 variable region, the replacement of at least one non-histidine codon with a histidine codon comprises a replacement at a position in the gene sequence of the immunoglobulin light chain 65 encoding CDR3 designed to express a histidine at the selected position between 105, 106, 108, 111 and a combination thereof. In one embodiment, the replacement is designed to express histidines at positions 105 and 106. In one embodiment, the replacement is designed to express histidines at positions 105 and 111. In one embodiment, the replacement is designed to express histidines at positions 105 and 108. In one embodiment, the replacement is designed to express histidines at positions 105, 108 and 111. In one embodiment, the replacement is designed to express histidines at positions 105, 106 and 108. In one embodiment, the 5 replacement is designed to express histidines at positions 106 and 108. In one embodiment, the replacement is designed to express histidines at positions 106 and 111. In one embodiment, the replacement is designed to express histidines at positions 108 and 111. In one embodiment, the replacement is designed to express histidines at positions 106, 108 and 111. In yet another embodiment, the replacement is designed to express histidines at positions. ones 106, 108 and 111. In one embodiment, the replacement is designed to express histidines at positions 10 105, 106 and 111. In one embodiment, the replacement is designed to express histidines at positions 105, 106, 108 and 111. The nucleic acid and amino acid sequences of the histidine substituted CDR3 regions are shown in the sequence alignment of FIG. 2 and is set forth in SEQ ID NO: 4-33. The nucleic acid and amino acid sequences of wild-type CDR3 (depicted in FIG. 2) are set forth in SEQ ID NO: 2 and 3, respectively. fifteen

In the embodiment in which the only rearranged human immunoglobulin light chain variable region is a Vκ3-20Jκ1 variable region, the replacement of at least one non-histidine codon with a histidine codon comprises a replacement at a position in the gene sequence of the immunoglobulin light chain encoding the CDR3 region that is designed to express a histidine in the position selected between 105, 106, 107, 109 and a combination thereof. In one embodiment, the replacement is designed to express histidines at positions 105 and 106. In one embodiment, the replacement is designed to express histidines at positions 105 and 107. In one embodiment, the replacement is designed to express histidines at positions 105 and 109. In one embodiment, the replacement is designed to express histidines at positions 106 and 109. In one embodiment, the replacement is designed to express histidines at positions 106 and 109. In one embodiment, the replacement is designed to expressing histidines at positions 107 and 109. In one embodiment, the replacement is designed to express histidines at positions 105, 106 and 107. In one embodiment, the replacement is designed to express histidines at positions 105, 107 and 109. In one embodiment, the replacement is designed to express histidines at positions 106, 108 and 111. In one embodiment, the replacement is designed to express histidines at positions 105 , 106 and 109. In another embodiment, the replacement is designed to express histidines at positions 105, 106, 107 and 109. The 30 nucleic acid and amino acid sequences of the example histidine substituted CDR3 regions are represented in the alignment of sequences of FIG. 12 and is set forth in SEQ ID NO: 76-79. The nucleic acid and amino acid sequences of wild-type CDR3 (depicted in FIG. 12) are set forth in SEQ ID NO: 74 and 75, respectively.

 35

The amino acid positions (105, 106, etc.) are based on a unique numbering described in Lefranc et al. (2003) Dev. Comp. Immunol 27: 55-77 and can also be seen at www.imgt.org.

In one aspect, the human VL gene segment is operably linked to a human or non-human leader sequence. In one aspect, the leader sequence is a non-human leader sequence. In a specific aspect, the non-human leader sequence is a mouse Vκ3-7 leader sequence. In a specific aspect, the leader sequence is operatively linked to a non-rearranged human VL gene segment. In a specific aspect, the leader sequence is operatively linked to a rearranged human VL / JL sequence. Thus, in a specific aspect, the only rearranged variable region gene sequence Vκ1-39 / Jκ5 or Vκ3-20 / Jκ1 comprising at least one substitution with histidine is operably linked to a mouse leader Vκ3-7 sequence. Four. Five

In one aspect, the VL gene segment is operably linked to an immunoglobulin promoter sequence. In one aspect, the promoter sequence is a human promoter sequence. In a specific aspect, the human immunoglobulin promoter is a human Vκ3-15 promoter. In a specific aspect, the promoter is operatively linked to a non-rearranged human VL gene segment. In a specific aspect, the promoter is operatively linked to a rearranged human VL / JL sequence. Thus, in a specific aspect, the only rearranged variable region gene sequence Vκ1-39 / Jκ5 or Vκ3-20 / Jκ1 comprising at least one substitution with histidine is operably linked to the human Vκ3-15 promoter.

In one aspect, the light chain locus comprises a leader sequence flanked at 5 '(with respect to the transcriptional direction of a VL gene segment) with a human immunoglobulin promoter and flanked at 3' with a human VL gene segment that is reorganizes with a human JL segment and encodes a variable domain of an inverse chimeric light chain comprising an endogenous nonhuman light chain (CL) constant region. In a specific aspect, the VL and JL gene segments are in the non-human Vκ locus and the non-human CL is a non-human Cκ (eg, mouse Cκ. In a specific aspect, the variable region sequence 60 is operably linked to the non-human constant region sequence, for example, the non-human Cκ gene sequence.

In one aspect, the light chain locus comprises a leader sequence flanked at 5 '(with respect to the transcriptional direction of a VL gene segment) with a human immunoglobulin promoter and flanked at 65 3' with a human variable region sequence rearranged (VL / JL sequence) and encodes a variable domain of a reverse chimeric light chain comprising a constant region of endogenous non-human light chain (C

In one aspect, the genetically modified non-human animal is a mouse and the mouse variable region locus is a κ light chain locus, and the κ light chain locus comprises an intrinsic mouse enhancer κ, a 3 'κ enhancer of mouse or both an intronic enhancer and a 3 'enhancer.

 fifteen

In one aspect, the non-human animal (for example, a rodent, for example, a rat or a mouse) comprises a non-functional lambda (λ) immunoglobulin light chain locus. In a specific aspect, the light chain locus A comprises a deletion of one or more locus sequences, in which the one or more deletions return to the light chain locus A unable to rearrange to form a light chain gene. In another aspect, all or substantially all of the VL gene segments of the light chain locus A are suppressed. In one aspect, the non-human animal 20 (eg, rodent, for example, mouse or rat) comprises a rearranged variable region sequence human immunoglobulin light chain comprising a substitution of at least one non-histidine codon with a histidine codon and lacks a functional non-rearranged immunoglobulin light chain region, for example, an endogenous non-rearranged light chain variable region . In one aspect, the rearranged histidine-substituted human immunoglobulin light chain variable region gene sequence replaces the endogenous non-rearranged immunoglobulin light chain variable region gene sequence.

In one aspect, the animal makes a light chain comprising a somatically mutated variable domain derived from a human variable region sequence comprising a substitution of at least one non-histidine codon with a histidine codon. In one aspect, the light chain comprises a mutated variable domain 30 somatically derived from a human variable region sequence comprising a substitution of at least one non-histidine codon with a histidine codon and a human Cκ region. In one aspect, the non-human animal does not express a κ light chain.

One skilled in the art will appreciate that, although the substitution or substitutions of at least one non-histidine moiety with a histidine moiety is genetically modified in the human immunoglobulin light chain variable region, due to somatic hypermutations, not all antibodies. that are generated in the genetically modified non-human animal will house that rest or histidine residues in the position or positions modified by genetic engineering. However, the generation of a large repertoire of antibodies in the non-human animal will allow the selection of antigen-specific antibodies generated in vivo that show high affinity for an antigen of interest that at the same time retain the histidine modifications introduced in the germ line and preferably have pH dependent antigen binding.

Thus, in one aspect, the animal retains at least one histidine amino acid introduced by replacing at least one non-histidine codon with a histidine codon in its variable region gene. In one aspect, animal 45 retains all or substantially all histidine substitutions in its somatically mutated light chain variable domain that were introduced into its variable region gene.

In one aspect, the genetically modified non-human animal described herein also comprises in its genome, for example, in its germ line, a non-rearranged immunoglobulin heavy chain variable region comprising VH gene segment sequences, DH and JH. In one aspect, the VH, DH and JH gene segment sequences are human VH, DH and JH gene segment sequences and the non-rearranged immunoglobulin heavy chain variable region is a human heavy chain variable region. In one aspect, the human VH, DH and JH gene segment sequences are operably linked to the non-human heavy chain constant region sequence. In one aspect, the non-human heavy chain constant region sequence is an endogenous non-human heavy chain constant region sequence. In one aspect, the human heavy chain gene segment sequences are in the endogenous non-human immunoglobulin heavy chain locus. In one aspect, the human immunoglobulin heavy chain variable region sequence comprised in a non-human animal also comprises a replacement of at least one non-histidine codon with a histidine codon. 60

In one aspect, the non-human animal described herein expresses an immunoglobulin light chain comprising a non-human light chain constant region sequence. In one aspect, the non-human animal expresses an immunoglobulin light chain comprising a constant region sequence of human light chain. 65

In one aspect, the non-human animal described herein expresses an immunoglobulin heavy chain comprising a non-human sequence selected from a CH1 sequence, a hinge sequence, a CH2 sequence, a CH3 sequence and a combination thereof. .

In one aspect, the non-human animal expresses an immunoglobulin heavy chain comprising a human sequence selected from a CH1 sequence, a hinge sequence, a CH2 sequence, a CH3 sequence and a combination thereof.

In the aspect in which the animal comprises a single variable region of rearranged immunoglobulin light chain comprising a replacement of at least one non-histidine codon with a histidine codon, the rearranged immunoglobulin light chain sequence in the germ line of the animal is in an endogenous non-human immunoglobulin light chain locus. In a specific aspect, the rearranged immunoglobulin light chain sequence comprising a substitution of at least one non-histidine codon with a histidine codon in the germ line of the animal replaces all or substantially all the chain V and J segment sequences. light endogenous non-human in the endogenous human immunoglobulin no 15 light chain locus.

In one aspect, the non-human animal comprises a replacement of endogenous VH gene segments with one or more human VH gene segments, in which the human VH gene segments are operably linked to a gene in the non-human CH region, so that the Non-human animal rearranges human VH gene segments 20 and expresses a heavy chain of reverse chimeric immunoglobulin comprising a human VH domain and a non-human CH. In one aspect, 90-100% of non-rearranged non-human VH gene segments are replaced with at least one non-rearranged human VH gene segment. In a specific aspect, all or substantially all (for example, 90-100%) of the endogenous non-human VH gene segments are replaced with at least one non-rearranged human VH gene segment. In one aspect, the replacement is with at least 19, at least 39 or at least 80 or 81 non-rearranged human VH gene segments. In one aspect, the replacement is with at least 12 functional non-rearranged human VH gene segments, at least 25 functional non-rearranged human VH gene segments, or at least 43 functional non-rearranged human VH gene segments. In one aspect, the non-human animal comprises a replacement of all non-human DH and JH segments with at least one unordered human DH segment and at least one unordered human JH segment. In one aspect, the non-human animal comprises a replacement of all non-human DH and JH segments with all non-rearranged human DH segments and all non-rearranged human JH segments.

A non-human animal, for example, a mouse, comprising in its genome, for example, in its germ line, a limited repertoire of variable regions of human immunoglobulin light chain, for example, a single variable region of light chain of rearranged human immunoglobulin (e.g., Vκ1-39 / Jκ5 or Vκ3-20 / Jκ1), with a replacement of at least one non-histidine codon with a histidine codon and a diverse repertoire of non-rearranged human VH, DH and JH segments It is capable of generating antigen-binding proteins encoded by heavy chain variable region sequences derived from various permutations of non-rearranged human VH, DH and JH segments, in which the VH, DH and JH segments present in the variable sequences of Heavy chain are derived from all or substantially all functional human VH, DH and JH segments present in the animal's genome. Various possibilities available for heavy chain variable domain sequences expressed in the cells, for example, B cells, of the genetically modified animals described herein are described (ie, derived from combinations of various segments V, D and J functional humans) in the US Application Publications No. 2011/0195454, 45 2012/0021409, 2012/0192300 and 2013/0045492. In various aspects, the human immunoglobulin light chain variable region rearranged sequence comprising the replacement or substitutions of at least one non-histidine codon with a histidine codon and the human immunoglobulin heavy chain variable region non-rearranged sequence are included in the germ line of the non-human animal.

 fifty

In one aspect, the non-human animal comprises a copy of one or both of the reordered sequence of human immunoglobulin light chain variable region comprising the substitution or substitutions of at least one non-histidine codon with a histidine codon and the sequence Non-rearranged heavy chain variable region of human immunoglobulin. In another aspect, the non-human animal comprises two copies of one or both of the reordered sequence of human immunoglobulin light chain variable region comprising substitution 55 or substitutions of at least one non-histidine codon with a histidine codon and the non-rearranged sequence of human immunoglobulin heavy chain variable region. Thus, the non-human animal may be homozygous or heterozygous for one or both of the rearranged human immunoglobulin light chain variable region sequence comprising the substitution or substitutions of at least one non-histidine codon with a histidine codon and the sequence Non-rearranged heavy chain variable region of human immunoglobulin. 60

In addition to genetically modified non-human animals that comprise in their genome an immunoglobulin light chain variable region gene sequence (for example, a single gene rearrangement of immunoglobulin light chain variable region gene) comprising the replacement of at least one codon Non-histidine with a histidine codon (for example, in light chain CDR3), also described herein are genetically modified non-human animals comprising an immunoglobulin light chain variable region gene sequence with one or more more additions / insertions of codon or histidine codons, so that the expressed variable domain comprises an additional amino acid or amino acids which, if not subjected to somatic hypermutation, is a histidine.

The genetically modified non-human animal comprising a human immunoglobulin light chain 5 variable region gene sequence with a substitution of at least one non-histidine codon with a histidine codon described herein can be selected from a group that It consists of a mouse, rat, rabbit, pig, bovine (for example, cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (for example, marmoset, Indian macaque). For non-human animals, in which suitable genetically modifiable ES cells are not readily available, methods other than those described herein are used to produce a non-human animal that comprises the genetic modification. Such methods include, for example, the modification of a genome of non-ES cells (for example, a fibroblast or an induced pluripotent cell) and the use of nuclear transfer to transfer the modified genome to a suitable cell, for example, an oocyte, and the gestation of the modified cell (for example, the modified oocyte) in a non-human animal under conditions suitable to form an embryo. fifteen

In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal, for example, of the Dipodoid or Muroidea superfamily. In one aspect, the genetically modified animal is a rodent. In one aspect, the rodent is selected from a mouse, a rat and a hamster. In one embodiment, the rodent is selected from the Muroidea superfamily. In one aspect, the genetically modified animal is from a family selected from Calomyscidae (for example, mouse-like hamsters), Cricetidae (for example, hamster, New World rats and mice, field mice), Muridae (mice and rats true gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, tail rats, Madagascar rats and mice), Platacanthomyidae (e.g., spiny lilies) and Spalacidae (e.g., mole rats, rats bamboo and zokors). In a specific embodiment, the genetically modified rodent is selected from a true mouse or rat (family Muridae), a gerbil, a spiny mouse and a crested rat. In one embodiment, the genetically modified mouse is from a member of the Muridae family. In one aspect, the animal is a rodent. In a specific embodiment, the rodent is selected between a mouse and a rat. In one embodiment, the non-human animal is a mouse.

 30

In a specific embodiment, the rodent is a mouse of a strain of C57BL selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr and C57BL / Ola. In another embodiment, the mouse is a strain of 129 selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / Svlm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, for example, Festing et al (1999) 35 Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836, see also, Auerbach et al (2000) Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines). In a specific embodiment, the genetically modified mouse is a mixture of a strain of 129 mentioned above and a strain C57BL / 6 mentioned above. In another specific embodiment, the mouse is a mixture of 129 strains mentioned above or a mixture of BL / 6 strains mentioned above. In a specific embodiment, the 129 strain of the mixture is a 129S6 strain (129 / SvEvTac). In another embodiment, the mouse is a strain of BALB, for example, strain BALB / c. In yet another embodiment, the mouse is a mixture of a strain of BALB and another strain mentioned above.

In one embodiment, the rodent is a rat. In one embodiment, the rat is selected from a Wistar rat, a strain 45 of LEA, a strain of Sprague Dawley, a strain of Fischer, F344, F6 and Dark Agouti. In one embodiment, the rat strain is a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6 and Dark Agouti.

Therefore, in one aspect, the genetically modified non-human animal is a rodent. In one aspect, the genetically modified non-human animal is a rat or a mouse. In one aspect, the animal is a mouse. Thus, in one aspect, a genetically modified mouse is described herein that comprises in its genome, for example, in its germ line, a single variable region of rearranged human immunoglobulin light chain comprising human VL and JL gene sequences , wherein the only rearranged human immunoglobulin light chain variable region comprises at least one non-histidine codon substitution with a histidine codon. In one aspect, the mouse lacks a functional non-rearranged immunoglobulin light chain variable region (for example, it lacks functional non-rearranged V and J gene segment sequences). In one aspect, the rearranged human immunoglobulin light chain variable region with substitution or histidine codon substitutions is the variable region Vκ1-39 / Jκ or Vκ3-20 / Jκ. In one aspect the segment segment J is selected from Jκ1, Jκ2, Jκ3, Jκ4 and Jκ5. In one aspect, the sequence of segment J is Jκ1 or Jκ5. In one embodiment, the replacement of at least one non-histidine codon with a histidine codon is in the nucleotide sequence encoding a CDR3 region. In one embodiment, in which the rearranged variable region sequence is the Vκ1-39 / Jκ5 sequence, the histidine substitution or substitutions are designed to be expressed at a selected position between 105, 106, 108, 111 and a combination of the same. In another embodiment, in which the rearranged variable region sequence is the Vκ3-20 / Jκ1 sequence, the substitution or substitutions with histidine are designed to be expressed in a selected position between 105, 106, 107, 109 and a combination from the same. In one embodiment, the immunoglobulin light chain variable region rearranged with codon or substituted histidine codons is operably linked to an endogenous mouse immunoglobulin constant region gene sequence (eg, the Cκ gene sequence). In one aspect, the mouse further comprises in its genome, for example, in its germ line, a heavy chain variable region of the non-rearranged immunoglobulin comprising V segments

This document also describes direction vectors for the generation of genetically modified non-human animals, for example, mice, which are described herein. In one aspect, a direction vector is described comprising, from 5 'to 3' in the transcriptional direction with reference to the sequences of the 5 'and 3' mouse homology arms of the vector, a mouse homology arm 5 ', a human or mouse immunoglobulin promoter 15, a human or mouse leader sequence, a human variable region selected from a rearranged human Vκ1-39Jκ5 or a rearranged human Vκ3-20Jκ1 and comprising a substitution of at least one non-codon of histidine with a histidine codon and a 3 'mouse homology arm. In one aspect, the 5 'and 3' homology arms direct the vector to a 5 'sequence with respect to an enhancer sequence that is present at 5' and close to the mouse Cκ gene. In another aspect, the direction vector 20 comprises a 5 'mouse homology arm followed by a selection cassette flanked by recombination sites, human or mouse immunoglobulin promoter, human or mouse leader sequence, a selected human variable region between a rearranged human Vκ1-39Jκ5 or a rearranged human Vκ3-20Jκ1 and comprising a replacement of at least one non-histidine codon with a histidine codon, followed by the 3 'mouse homology arm comprising mouse enhancers and a region constant (Cκ sequences). 25

A selection cassette is a nucleotide sequence inserted into an address construct to facilitate the selection of cells (eg, ES cells) that have integrated the construct of interest. A series of suitable selection cassettes are known in the art. Generally, a selection cassette allows positive selection in the presence of a particular antibiotic (e.g., Neo, Hyg, Pur, CM, Spec, etc.). In addition, a selection cassette may be flanked by recombination sites, allowing deletion of the selection cassette after treatment with recombinant enzymes. LoxP and Frt recombination sites are commonly used, recognized by Cre and Flp enzymes, respectively, but others are known in the art.

In one aspect, the promoter is a human immunoglobulin variable region gene segment promoter. In a specific aspect, the promoter is a human Vκ3-15 promoter. In one aspect, the leader sequence is a mouse leader sequence. In a specific aspect, the mouse leader sequence is a mouse Vκ3-7 leader sequence. Example aspects of the address vectors are presented in FIG. 8B and 14B.

In one aspect, a direction vector is as described above, but instead of the 5 'mouse homology arm 40 the human or mouse promoter is flanked at 5' with a site-specific recombinase recognition site (SRRS , from the English site-specific recombinase recognition site) and instead of the 3 'homology arm the human VL region is flanked at 3' with an SRRS.

Methods for producing genetically modified non-human animals (eg rodents, for example, mice or rats) described herein are also described herein. In one aspect, the method of producing a genetically modified non-human animal described herein uses a direction vector, made using VELOCIGENE® technology, introducing the construction into ES cells and introducing clones of targeted ES cells into an embryo of mouse using VELOCIMOUSE® technology, as described in the Examples. Histidine modifications can be made to the targeting vector 50 using a variety of molecular biology techniques, for example, site-directed mutagenesis or de novo DNA synthesis. Upon termination of the gene address, ES cells of genetically modified non-human animals are screened to confirm the successful incorporation of the exogenous nucleotide sequence of interest or expression of exogenous polypeptide. Numerous techniques are known to those skilled in the art, and include (but are not limited to) Southern blotting, long PCR, quantitative PCT (e.g., real-time PCR using TAQMAN®), fluorescence in situ hybridization, Northern blotting. , flow cytometry, Western analysis, immunocytochemistry, immunohistochemistry, etc. In one example, non-human animals (eg, mice) that carry the genetic modification of interest can be identified by detecting the loss of the mouse allele and / or the gain of the human allele using a modification of the allele assay described in Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression 60 analysis, Nature Biotech. 21 (6): 652-659. Other assays that identify a specific sequence of nucleotides or amino acids in genetically modified animals are known to those skilled in the art.

Thus, in one aspect, the method of generating genetically modified non-human animals comprises replacing an immunoglobulin light chain variable region gene sequence in the animal with a human immunoglobulin light chain variable region gene sequence (comprising gene segments V

 5

In one aspect, the method of generating genetically modified non-human animals described herein comprises replacing an immunoglobulin light chain variable region gene sequence in the animal with a single rearranged light chain variable region gene sequence. human immunoglobulin comprising sequences of human VL and JL segments, in which the only rearranged gene sequence of human immunoglobulin variable region comprises a substitution of at least one non-histidine codon with a histidine codon. In one aspect, the substitution is in a CDR codon. In one aspect, the substitution is one, two, three, four or more CDR3 codons. In one aspect, the unique human immunoglobulin light chain variable region rearranged gene sequence is based on the human germ line light chain variable region rearranged sequence selected between Vκ1-39Jκ5 and Vκ3-20Jκ1. Thus, in one embodiment, in which the only rearranged gene region of the human immunoglobulin 15 light chain variable region is derived from Vκ1-39Jκ5, the replacement of at least one non-histidine codon with a histidine codon is designed to express a histidine in the selected positions between 105, 106, 108, 111 and a combination thereof. In one embodiment, in which the only rearranged gene region of the immunoglobulin light chain variable region is derived from Vκ3-20k1, the replacement of at least one non-histidine codon with a histidine codon is designed to express a histidine in one position. selected from 105, 106, 107, 109 and a combination thereof.

In another aspect, the method of generating a non-human animal described herein (i.e., comprising a genetically modified immunoglobulin light chain locus described herein) comprises modifying a genome of an animal. non-human to suppress or return non-functional 25 and V-segment segments of endogenous immunoglobulin light chain in an immunoglobulin light chain locus and to locate in the genome a single rearranged gene sequence of human light chain variable region comprising a substitution of al minus a non-histidine codon with a histidine codon. In one aspect, the method results in a genetically modified non-human animal comprising a population of B cells enriched for antibodies that exhibit pH-dependent binding to an antigen of interest.

In some aspects, the genetically modified non-human animal generation methods described herein comprise replacing an immunoglobulin light chain variable region gene sequence with a human immunoglobulin 35 light chain variable region gene sequence comprising the substitution or substitutions of at least one non-histidine codon with a histidine codon in the animal, which also comprises replacing an endogenous non-human immunoglobulin heavy chain variable region gene sequence with a heavy chain variable region gene sequence of human immunoglobulin comprising at least one of each or a repertoire of human VH, DH and JH sequences as described above. In one aspect, in order to generate a non-human animal that comprises an endogenous immunoglobulin light chain variable region gene sequence replacement human immunoglobulin light chain variable region gene sequence comprising a substitution of at least one codon non-histidine with a histidine codon and an endogenous non-human immunoglobulin heavy chain variable region gene sequence replacement with a human immunoglobulin heavy chain variable region gene sequence, the animal with the region gene sequence replacement The light chain variable is reproduced to an animal with a heavy chain variable region gene sequence replacement.

The inventors describe herein non-human animals engineered (for example, rodents, for example, rats or mice) that express antigen-binding proteins, for example, 50 antibodies, which comprise a universal light chain, for example , a human universal light chain (for example, a light chain derived from a single variable region of rearranged human immunoglobulin light chain) comprising one or more histidine modifications, in which the antigen binding proteins have an antigen binding pH dependent of a target antigen. Animals are engineered to include a light chain CDR3 comprising one or more histidine modifications. In various aspects, light chain CDR3 comprises two, three or four or more histidine residues in a cluster.

In one aspect, a genetically modified non-human animal (eg, a mouse or a rat) comprising a population of B cells characterized by an enhanced presence of histidines in immunoglobulin light chains is described herein, by For example, immunoglobulin variable domains, for example, immunoglobulin CDR, compared to a wild-type animal. In one aspect, the potentiation of the presence of histidine is approximately 2 to 4 times. In one aspect, histidine potentiation is about 2 to 10 times.

 65

In one aspect, a genetically modified non-human animal comprising a population of antigen-specific antibodies expressing a histidine moiety or residues as a result of codon modifications in the chain variable region gene sequence is described herein. light and shows pH dependent binding to the target antigen. In one aspect, these animals comprise a population of B cells that are enriched for antibodies, for example, antigen-specific antibodies, which show 5 pH-dependent binding properties (e.g., decrease in dissociative half-life (t

Therefore, an antigen-binding protein, generated in genetically modified non-human animals described herein, is described herein, in which the antigen-binding protein shows pH-dependent antigen binding. In one aspect, the antigen binding protein is an antibody, for example, antigen specific antibody. In one aspect, the antibody comprises a light chain comprising a human light chain variable domain derived from a rearrangement of human immunoglobulin light chain variable gene segments in which at least one non-histidine codon was replaced by a codon of histidine in the germ strain gene sequence and in which the antibody retains at least one substitution with histidine in its expressed human light chain variable domain. In one aspect, the antibody comprises a light chain comprising a human light chain variable domain derived from a single rearranged human light chain variable region gene sequence, in which the light chain variable region rearranged gene sequence only comprises one substitution of at least one non-histidine codon with a histidine codon and in which the antibody retains at least one substitution with histidine in its expressed light chain variable domain. In one aspect, the antibody comprises a light chain derived from a human Vκ1-39Jκ5 or Vκ3-20Jκ1 rearrangement, in which the human Vκ1-39Jκ5 or Vκ3-20Jκ1 gene sequence comprises a substitution of at least one non-histidine codon with a histidine codon and in which the antibody retains at least one substitution with histidine in its expressed light chain variable domain. In some aspects, the antibody retains all or substantially all histidine substitutions in its expressed light chain variable domain. In one aspect, the substitution is three non-histidine codons with three histidine codons in the nucleotide sequence encoding CDR3 of the light chain variable region gene sequence and the antibody retains all three histidine substitutions in its variable domain of Light chain expressed. In one aspect, the substitution is four non-histidine codons with four 40 histidine codons in the nucleotide sequence encoding CDR3 of the light chain variable region gene sequence and the antibody retains three or four histidine substitutions in its domain Light chain variable expressed.

In one aspect, the antibody light chain additionally comprises a non-human light chain constant region amino acid sequence, for example, an endogenous light chain constant region amino acid sequence. In addition, the antibody, for example, an antigen specific antibody, generated in a genetically modified non-human animal described herein also comprises a heavy chain comprising a human heavy chain variable domain derived from a rearrangement of segments V , D and J heavy human chain. Human heavy chain segments V, D and J may be selected from a repertoire of human heavy chain segments present in the endogenous nonhuman heavy chain locus, for example, at least one functional segment V, at least one functional segment D and at least one functional J segment, for example, up to a complete repertoire of functional human V, D and J segments. Possible example rearrangements of human heavy chain variable segments can be obtained from a list of functional human V, D and J segments in the IMGT database and from US Application Publications 55. No. 2011/0195454, 2012/0021409, 2012/0192309 and 2013/0045492. In addition, in one aspect, the antibody heavy chain comprises a non-human heavy chain constant region amino acid sequence, for example, an endogenous nonhuman heavy chain constant region amino acid sequence. In one aspect, the endogenous non-human heavy chain constant region comprises domains CH1, hinge, CH2 and CH3. In one aspect, the antibody is an IgG, IgE, IgD, IgM or IgA isotype. 60

Therefore, in one aspect, a binding protein generated in the genetically modified non-human animals described herein is described herein, wherein the binding protein comprises a reverse chimeric light chain comprising (a ) a variable light chain domain derived from a human Vκ1-39Jκ5 rearrangement comprising a substitution of at least one non-histidine codon with a histidine codon, in which the light chain retains at least one substitution with histidine in its domain expressed light chain variable and (b) a non-human light chain constant region amino acid sequence, for example, of mouse, in which the light chain is associated with an inverse chimeric heavy chain comprising (a) a variable domain heavy chain derived from a reordering of segments V, D and J human, in which segments V, D and J are selected from a repertoire of segments V, D and J hu hands present in the animal and (b) a non-human heavy chain constant region amino acid sequence, for example, mouse. In one aspect, the repertoire of human segments V, D and J comprises at least one functional segment V, at least one functional segment D and at least one functional segment J, for example, up to a complete repertoire of segments V, D and J functional humans. In one aspect, heavy chain and light chain constant domains are constant regions of endogenous heavy and light chain. In one aspect, the heavy and light chain variable domains are somatically mutated domains. In one aspect, the somatically mutated light chain domain 10 retains at least one substitution with histidine introduced into the germ line sequence. In some aspects, the somatically mutated light chain domain retains all or substantially all histidine substitutions introduced into the germ line sequence. In one aspect, the antigen binding protein shows pH dependent antigen binding properties.

 fifteen

In another aspect, a binding protein generated in the genetically modified non-human animals described herein is described herein, wherein the binding protein comprises a reverse chimeric light chain comprising (a) a domain light chain variable derived from a human Vκ3-20Jκ1 rearrangement comprising a substitution of at least one non-histidine codon with a histidine codon, in which the light chain retains at least one substitution with histidine in its domain 20 chain variable expressed light weight and (b) a non-human light chain constant region amino acid sequence, for example, of mouse, in which light chain is associated with an inverse chimeric heavy chain comprising (a) a derived heavy chain variable domain of a rearrangement of human segments V, D and J, in which segments V, D and J are selected from a repertoire of human segments V, D and J present it is in the animal and (b) a non-human heavy chain constant region amino acid sequence, for example, of mouse. In one aspect, the repertoire of human segments V, D and J comprises at least one functional segment V, at least one functional segment D and at least one functional segment J, for example, up to a complete repertoire of segments V, D and J functional humans. In one aspect, the heavy chain and light chain constant regions are endogenous heavy and light chain constant regions. In one aspect, the heavy and light chain variable domains are somatically mutated domains. In one aspect, the somatically mutated light chain domain 30 retains at least one histidine substitution introduced into the germ line sequence. In some aspects, the somatically mutated light chain domain retains all or substantially all histidine substitutions introduced into the germ line sequence. In one aspect, the antigen binding protein shows pH dependent antigen binding properties.

 35

In one aspect, a B cell of the transgenic animal described herein is also described herein, which comprises in its germ line a histidine modified human light chain variable region sequence, for example, a single sequence human light chain variable region rearrange modified with histidine, which is described herein and expresses an antigen-binding protein described herein. In one aspect, the antigen-binding protein, for example, an antibody, expressed in cell B, retains at least one histidine residue introduced into the germ line and shows pH-dependent antigen-binding properties. In some aspects, the antigen-binding protein, for example, an antibody, expressed in cell B, retains all or substantially all histidine residues introduced into the germ line and shows pH-dependent antigen-binding properties.

 Four. Five

In various aspects, the genetically modified non-human animal described herein comprises a human light chain variable region gene sequence, for example, a single rearranged human light chain variable region gene sequence (e.g., the sequence Vκ1-39Jκ5 or Vκ3-20Jκ1) comprising a substitution of at least one non-histidine codon with a histidine codon (or an addition of a histidine codon in the germ line sequence). These additions or substitutions result in a non-human animal comprising a population of B cells enriched for antigen binding proteins with pH dependent binding properties for their antigens. In one aspect, antigen-binding proteins, for example, antibodies, generated in non-human animals described herein in response to antigen stimulation show a pH-dependent antigen binding while exhibiting high affinity. by the antigen at neutral pH, for example, at a pH between about 7.0 and about 8.0, for example, a pH between about 7.0 and about 7.4, for example, between about 7 , 2 and about 7.4, for example, a physiological pH. In one aspect, the affinity of the antigen-binding protein for its antigen, expressed as a dissociation constant (KD) at a neutral pH is less than 10-6 M, for example, less than 10-8 M, for example , less than 10-9 M, for example, less than 10-10 M, for example, less than 10-11 M, for example, less than 10-12 M. 60

In one aspect, an antigen-binding protein, for example, an antibody, generated in the genetically modified non-human animal described herein, shows a reduced binding to its antigen at acidic pH (for example, at a pH of 6.0 or less, for example, a pH between about 5.0 and about 6.0, a pH between about 5.75 and about 6.0, for example, a pH of 65 endosomal or lysosomal compartments) compared to a neutral pH. In one aspect, the antigen-binding protein, for example, the antibody, generated in the genetically modified non-human animal described herein, does not exhibit any antigen binding at acidic pH, while retaining antigen binding. at neutral pH. In one aspect, an antigen-binding protein generated by the genetically modified non-human animal described herein, has a decrease in dissociative half-life (t

Kinetic parameters, such as dissociation equilibrium constants (KD) and dissociative half-lives (t1 / 2), can be calculated from the constant kinetic velocity as: KD (M) = kd / ka; and t1 / 2 (min) = ln2 / (60 * kd). twenty

In one aspect, the antigen-binding protein, for example, an antibody, generated in the genetically modified non-human animals described herein, has an increased binding to the FcRn molecule. As described above, FcRn is a receptor present inside the endosomal compartment that is able to bind to immunoglobulins at an acidic pH and recycle them back to the surface. The detection of antibody molecules in the genetically modified non-human animals described herein presents a unique opportunity to select antibodies with three beneficial parameters: high affinity for an antigen, pH dependent antigen binding (with an antigen binding weaker at acidic pH) and an increased binding to FcRn.

 30

In one aspect, a genetically modified non-human animal described herein comprises a population of B cells in response to an antigen that produces and is enriched in antigen-binding proteins, for example, antibodies, which, when reformatted in therapeutic products, they have an increased half-life in serum after the administration of a therapeutic dose to a subject on a population of equivalent B cells produced in response to the same antigen in non-human animals that do not comprise a modification or modifications with histidine in their gene sequences of human light chain variable region. Thus, in one aspect, an antigen-binding protein, for example, an antibody, produced in response to an antigen of interest in a genetically modified non-human animal described herein, when reformatted into therapeutic products, it has an increased serum half-life after the administration of a therapeutic dose to a subject during a serum half-life of an antigen binding protein 40 (when reformatted into a therapeutic product and administered at the same therapeutic dose) with respect to which it was produced in response to the same antigen in a non-human animal that did not comprise any modification or modifications with histidine in its human light chain variable region gene sequence. In some aspects, the increase in serum half-life is about 2 times, for example, about 5 times, for example, about 10 times, for example, about 15 times, for example, about 20 times or more.

In one aspect, a pluripotent, induced pluripotent or totipotent cell derived from a non-human is described herein as described herein. In a specific aspect, the cell is an embryonic stem cell (ES). fifty

In one aspect, a tissue derived from a non-human animal is provided as described herein. In one aspect, the tissue is derived from the spleen, lymph nodes or bone marrow of a non-human animal as described herein.

 55

In one aspect, a core derived from a non-human animal is described herein as described herein. In one aspect, the nucleus is of a diploid cell that is not a B cell.

In one aspect, a non-human cell that is isolated from a non-human animal (for example, a rodent, for example, a mouse or a rat) is described herein as described herein. In one aspect, the cell is an ES cell. In one embodiment, the cell is a lymphocyte. In one aspect, the lymphocyte is a B cell. In one aspect, the B cell expresses a chimeric heavy chain comprising a variable domain derived from a human gene segment; and a light chain derived from a rearranged human Vκ1-39 / J sequence with a substitution of at least one non-histidine codon with a histidine codon, a rearranged human Vκ3-20 / J sequence with a replacement of at least one non-codon histidine with a histidine codon or a combination thereof and further comprising a substitution of at least one amino acid encoded in the germ line for a histidine; wherein the heavy chain variable domain is fused with a constant region of human or non-human heavy chain and the light chain variable domain is fused with a constant region of human or non-human light chain.

In one aspect, a hybridoma is described herein, in which the hybridoma is made with a B 5 cell of a non-human animal as described herein. In a specific aspect, cell B is from a mouse as described herein that has been immunized with an immunogen comprising an epitope of interest, and cell B expresses a binding protein that binds to the epitope of interest, the binding protein has a somatically mutated human heavy chain variable domain and a mouse CH, and has a human variable light chain domain derived from a rearranged human Vκ1-39Jκ5 with a replacement of at least 10 a non-histidine codon with a histidine codon or a rearranged human Vκ3-20Jκ1 with a substitution of at least one non-histidine codon with a histidine codon and a mouse CL, wherein the human light chain domain comprises a substitution of at least one amino acid encoded in the germ line with a histidine.

Also described herein is a cell that expresses an antigen-binding protein generated in nonhuman animals described herein. In one aspect, the cell is selected from CHO, COS, 293, HeLa and a retinal cell that expresses a viral nucleic acid sequence (for example, a PERC.6 ™ cell).

In one aspect, a non-human embryo is described herein, in which the embryo comprises a donor ES cell derived from a non-human animal as described herein.

The non-human animals described herein are useful for generating B cells that express antibodies that have histidines in a CDR3. An animal that locates histidines in a CDR3 is useful for producing antibodies in general, and, in particular, is useful for developing antibodies that bind to a target with sufficient affinity at, or around, a neutral pH, but that either they do not bind or bind weaker to the same target at an acidic pH.

The non-human animal is useful for generating variable regions of antibodies that can be used to make, for example, human therapeutic binding proteins that bind to their targets by human variable immunoglobulin domains comprising histidines in a CDR3. Altered binding at a lower pH in some circumstances will allow faster renewal because the therapeutic agent will bind to a target on the surface of a cell, will be internalized in an endosome and will more easily or more rapidly dissociate from the target in the endosome, so that the therapeutic agent can be recycled to bind to another target molecule (for example, on another cell or the same cell). In some circumstances, this will result in the ability to dose the therapeutic agent at a lower dose or dose the therapeutic agent less frequently. This is particularly useful when it is not desirable to dose frequently or to administer above a certain dose, for safety or toxicity reasons. As a result, the serum half-life of the therapeutic antibody will increase when administered to a subject.

 40

The non-human animal, for example, rodent, for example, mouse or rat, is useful in a method for increasing the number of B cells in an animal that has a variable region of antibody that has a CDR3 with one or more histidines in the same. The non-human animal is useful for generating antibody sequences that will exhibit pH dependent antigen binding. The non-human animal is useful for generating a greater number of antibody sequences, as a result of a single immunization, in which the antibodies will exhibit pH-dependent antigen binding.

Antigen binding proteins and methods to generate them

In one aspect, methods for generating binding proteins to 50 human antigens, for example, antibodies, which exhibit pH-dependent antigen binding, from the genetically modified non-human animals described herein are also described. present document with conventional methods used in the art.

Several techniques for the production of antibodies have been described. For example, in several aspects 55 chimeric antibodies are produced in mice as described herein. Antibodies can be isolated directly from B cells of an immunized mouse (for example, see US 2007 / 0280945A1) and / or the B cells of the immunized mouse can be used to produce hybridomas (Kohler and Milstein, 1975, Nature 256: 495 -497). DNA encoding antibodies (human heavy and / or light chains) from non-human animals as described herein is easily isolated and sequenced using conventional techniques. The hybridoma and / or B cells derived from non-human animals as described herein serve as a preferred source of said DNA. Once isolated, the DNA can be located in expression vectors, which are then transfected into host cells that otherwise do not produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA can also be modified, for example, by replacing the coding sequence with constant domains of human heavy and light chain instead of non-human sequences. Thus, once antibody nucleic acid sequences with the desired characteristics are determined, for example, affinity, epitope, pH dependent antigen binding, etc., and non-human constant region gene sequences are replaced with a desired human constant region sequence to generate a completely human antibody that contains a non-IgM isotype, for example, IgG1, IgG2, IgG3 or IgG4.

 5

Therefore, in one aspect, a method of generating an antibody having pH dependent antigen binding properties comprising generating a non-human animal (eg, a mouse) as described herein is described herein. document, immunize a mouse with an antigen of interest, allow a non-human animal to mount an immune response to the antigen and select in the non-human animal an antigen-specific antibody that has pH-dependent antigen binding properties, 10 for example, the weaker binding to the antigen at an acidic pH than at a neutral one.

Also described herein are methods for producing multi-specific antigen binding proteins, for example, bispecific antigen binding proteins. These are molecules capable of binding to more than one epitope with high affinity. Advantages of the invention include the ability to properly select 15 high-binding heavy chain immunoglobulin chains (eg, matured affinity) each of which will be associated with a single light chain. In addition, the advantages of the invention include the ability to generate a multi-specific antigen-binding protein, for example, bispecific, which exhibits pH-dependent antigen binding.

 twenty

Due to the dual nature of bispecific antibodies (i.e., they may be specific for different epitopes of a polypeptide or they may contain specific antigen binding domains for more than one target polypeptide, see, for example, Tutt et al., 1991, J. Immunol. 147: 60-69; Kufer et al, 2004, Trends Biotechnol 22: 238-244), offer many useful advantages for therapeutic application. For example, bispecific antibodies can be used for redirected cytotoxicity (for example, to destroy tumor cells), such as a vaccine adjuvant, to deliver thrombolytic agents to clots, to convert enzyme-activated prodrugs to a target site (e.g., a tumor), to treat infectious diseases, to direct immune complexes to cell surface receptors or to deliver immunotoxins to tumor cells.

The bispecific antibodies described herein can also be used in several therapeutic and non-therapeutic and / or diagnostic test methods, such as, enzyme immunoassays, two-site immunoassays, in vitro or in vivo immunodiagnostics of various diseases ( for example, cancer), competitive binding assays, direct and indirect sandwich assays and immunoprecipitation assays. Other uses for bispecific antibodies will be apparent to those skilled in the art.

 35

Several techniques for producing bispecific antibody fragments from recombinant cell culture have been published. However, the synthesis and expression of bispecific binding proteins have been problematic, partly due to problems associated with the identification of a suitable light chain that can be associated and expressed with two different heavy chains and partly due to isolation problems. In various aspects, the compositions and methods described herein provide the advantage of 40 full-length bispecific antibodies that do not require any modification or special modifications to maintain the traditional immunoglobulin structure by increasing the stability / interaction of the components. In several aspects, such modification or modifications have proved cumbersome and are an obstacle to the development of bispecific antibody technology and its potential use in the treatment of human diseases. Thus, in various aspects, by providing a structure of natural immunoglobulin (ie, full length) having the added property of multiple specificities, full length bispecific antibodies maintain their critical effector functions that were lacking. previous bispecific fragments and additionally provide therapeutic agents that demonstrate the important pharmacokinetic parameter of a longer half-life.

 fifty

The methods and compositions described herein allow a genetically modified mouse to select, through otherwise natural processes, a suitable light chain that can be associated and expressed with more than one heavy chain, including heavy chains that are mutated. somatically (for example, affinity matured), in which the light chain additionally gives the antigen-binding protein its pH-dependent antigen-binding property. The heavy and light chain human variable region sequences of suitable B cells from immunized mice as described herein that express matured affinity antibodies having reverse chimeric heavy chains (ie, human variable and mouse constant) can identified and cloned in phase into an expression vector with a suitable human constant region gene sequence (eg, a human IgG1). Two such constructs can be prepared, in which each construct encodes a variable domain of human heavy chain that binds to a different epitope. One of the human light chain variable regions (e.g., human Vκ1-39Jκ5 or human Vκ3-20Jκ1), which comprises a replacement of at least one non-histidine codon with a histidine codon, can be fused in phase with a gene of suitable human light chain constant region (for example, a human κ constant gene). These three heavy and light all-human constructions can be located in a cell suitable for expression. The cell will express two main species 65: a homodimeric heavy chain with the identical light chain and a heterodimeric heavy chain with the identical light chain. To allow easy separation of these major species, one of the heavy chains is modified to omit a protein A binding determinant, resulting in a differential affinity of a homodimeric binding protein with respect to a heterodimeric binding protein. Compositions and methods that address this problem are described in USSN 12 / 832,838, filed June 25, 2010, entitled "Readily Isolated Bispecific Antibodies with Native Immunoglobulin Format", published 5 as US 2010 / 0331527A1. Once the species comprising the heterodimeric heavy chain with an identical light chain is selected, this bi-specific antigen binding protein can be screened to confirm the retention of its pH dependent antigen binding property.

In one aspect, an epitope binding protein is described herein as described herein, in which human light chain and heavy chain variable region sequences are derived from animals described herein. document that have been immunized with an antigen comprising an epitope of interest.

In one aspect, an epitope binding protein is provided comprising a first and a second polypeptide, the first polypeptide comprising, from the N-terminus to the C-terminus, a first epitope binding region that selectively binds to a first epitope, followed by a constant region comprising a first CH3 region of a human IgG selected from IgG 1, IgG2, IgG4 and a combination thereof; and, a second polypeptide comprising, from end N to end C, a second epitope binding region that selectively binds to a second epitope, followed by a constant region comprising a second CH3 region of a human IgG 20 selected from IgG 1, IgG2, IgG4 and a combination thereof, wherein the second CH3 region comprises a modification that reduces or eliminates the binding of the second CH3 domain to protein A. Various modifications of this type are described, for example, in the US Application Publications No. 2010/0331527 and 2011/0195454.

 25

A method for the production of an epitope binding protein that binds to more than one epitope and has a pH dependent epitope binding property is to immunize a first mouse according to the invention with an antigen comprising a first epitope of of interest, in which the mouse comprises (1) an endogenous immunoglobulin light chain variable region locus that does not contain an endogenous mouse light chain variable region gene sequence that is capable of rearranging and forming a light chain, in the that in the endogenous mouse immunoglobulin light chain variable region locus 30 is a single rearranged human light chain variable region operatively linked to the mouse endogenous light chain constant region gene, and the rearranged human light chain variable region is selects between a human Vκ1-39Jκ5 and a human Vκ3-20Jκ1 comprising a substitution of at least one non-histidine codon with a Histidine codon and (2) the endogenous mouse VH gene segments have been replaced in whole or in part with human VH 35 gene segments, so that the immunoglobulin heavy chains produced by the mouse are only or substantially heavy chains comprising human variable domains and mouse constant domains. When immunized, such a mouse will produce a reverse chimeric antibody, comprising only one of two human light chain variable domains (for example, one of human Vκ1-39Jκ5 or human Vκ3-20Jκ1, for example, comprising a substitution of at least one amino acid with a histidine). Usually, at least some of the substituted histidine residues introduced into the germ line sequence in the reverse chimeric antibody will be conserved. Once a B cell is identified that encodes a heavy chain variable domain that binds to the epitope of interest and expresses an antibody that has antigen-dependent properties dependent on, the nucleotide sequence of the heavy chain variable region ( and, optionally, the variable region of the light chain) can be recovered (for example, by PCR) and can be cloned in a phase expression construct with a suitable human immunoglobulin heavy chain constant region sequence. This process can be repeated to identify a second heavy chain variable domain that binds to a second epitope and a second heavy chain variable region gene sequence can be recovered and cloned in a phase expression vector to a second constant region sequence of suitable human immunoglobulin heavy chain. The first and second immunoglobulin 50 constant domains encoded by the constant region gene sequence may be the same or different from the isotype and one of the immunoglobulin constant domains (but not the other) may be modified as described herein or in the US 2010 / 0331527A1 and an epitope binding protein can be expressed in a suitable cell and isolated based on its differential affinity for protein A compared to a homodimeric epitope binding protein, for example, as described in US document. 55 2010 / 0331527A1.

Therefore, in various aspects, after DNA isolation and the selection of the first and second nucleic acid sequences encoding the first and second human heavy chain variable domains having the desired specificities / affinities and a third nucleic acid sequence which encodes a human light chain domain (a rearranged germ line sequence or a light chain sequence isolated from a non-human animal as described herein) and comprises a replacement of at least one non-histidine codon with a Histidine codon, the three nucleic acid sequences encoding the molecules are expressed to form the bispecific antibody using recombinant techniques that are widely available in the art. Frequently, the expression system of choice will involve an expression vector of 65 mammalian and host cells so that the bispecific antibody is properly glycosylated (for example, in the case of bispecific antibodies comprising antibody domains that are glycosylated). However, molecules can also be produced in prokaryotic expression systems. Normally, the host cell is transformed with DNA encoding both the first human heavy chain variable domain, the second human heavy chain variable domain, the human light chain domain in a single vector or in independent vectors. However, it is possible to express the first human heavy chain variable domain, the second human heavy chain variable domain and the human light chain domain (the components of bispecific antibodies) in independent expression systems and couple the in vitro expressed polypeptides . In various aspects, the human light chain domain derives from a germ line sequence, except for the replacement of at least one non-histidine codon with a histidine codon, for example, in a CDR codon. In various aspects, the human light chain domain comprises no more than one, no more than 10 two, no more than three, no more than four or no more than five somatic hypermutations within the light chain variable sequence of the light chain domain . In some aspects, somatic hypermutations do not alter the presence of at least one histidine residue introduced into the sequence of germline strain of light chain variable region.

 fifteen

In various aspects, the acid or nucleic acids (eg, cDNA or genomic DNA) encoding the two heavy chains and the only human light chain with a replacement of at least one non-histidine codon with a histidine is inserted into a replicable vector for additional cloning (amplification of DNA) and / or for expression. Many vectors are available and generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a termination sequence of the transcription. Each component can be selected individually or based on the choice of a host cell or other experimentally determined criteria. Several examples of each component are known in the art.

Expression and cloning vectors generally contain a promoter that is recognized by the host organism and operably binds to the nucleic acid sequences encoding each or all components of the bispecific antibody. A large number of promoters recognized by a variety of potential host cells are well known. These promoters are operably linked to the DNA encoding the bispecific antibody by removing the promoter from the original DNA by restriction enzyme digestion and inserting the promoter sequence isolated in the vector. 30

Expression vectors used in eukaryotic host cells (yeast cells, fungi, insects, plants, animals, humans or nucleated cells of other multicellular organisms) may also contain sequences necessary for termination of transcription and to stabilize mRNA. Such sequences are usually available from the 5 'and, occasionally 3', untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain transcribed nucleotide segments as polyadenylated fragments in the untranslated portion of the mRNA that encodes the components of bispecific antibodies. Expression vectors suitable for various embodiments include those that provide transient expression in mammalian cells of DNA encoding the bispecific antibody. In general, transient expression involves the use of an expression vector that is capable of efficiently replicating in a host cell, so that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector. Transient expression systems, comprising a suitable expression vector and a host cell, allow convenient positive identification of cloned DNA-encoded polypeptides, as well as for the rapid detection of bispecific antibodies having desired binding specificities / affinities or characteristics. of gel migration desired with respect to parental antibodies having homodimers of the first or second human heavy chain variable domains.

In various aspects, once the DNA encoding the components of the bispecific antibody are assembled into the desired vector or vectors as described above, they are introduced into a host cell suitable for expression and recovery. Transfection of the host cells can be carried out using conventional techniques known in the art appropriate for the selected host cell (for example, electroporation, nuclear microinjection, fusion of bacterial protoplasts with intact cells or polycations, for example, polybrene, polynithine, etc.) .

 55

In various aspects, a host cell is chosen that is better suited to the expression vector containing the components and allows more efficient and favorable production of the bispecific antibody species. Exemplary host cells for expression include those of prokaryotes and eukaryotes (single cell or multi-cell), bacterial cells (e.g., E. coli strains, Bacillus spp., Streptomyces spp., Etc.), cells of mycobacteria, fungal cells, yeast cells (for example, S. cerevisiae, S. pombe, P. pastoris, P. 60 methanolica, etc.), plant cells, insect cells (for example, SF-9, SF- 21, insect cells infected with baculovirus, Trichoplusia ni, etc.), non-human animal cells, human cells or cell fusions such as, for example, hybridomas or quadromas. In various aspects, the cell is a human, monkey, ape, hamster, rat or mouse cell. In various aspects, the cell is eukaryotic and is selected from the following cells: CHO (for example, CHO K1, DXB-11 CHO, Veggie-CHO), COS (for example, COS-7), 65 retinal cells, Vero, CV1, kidney (for example, HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (for example, BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, 3A BRL cells, HT1080 cells, myeloma cells, tumor cells and a cell line derived from a cell mentioned above. In various aspects, the cell comprises one or more viral genes, for example, a retinal cell that expresses a viral gene (for example, a PER.C6 ™ cell). 5

The mammalian host cells used to produce the bispecific antibody can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for Cultivate host cells. The media may be supplemented as necessary with 10 hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES ), nucleosides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN ™), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) and glucose or an equivalent energy source. Any other supplement may also be included in the appropriate concentrations, as is known to those skilled in the art. The culture conditions, such as temperature, pH and the like, are, in various embodiments, those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

The bispecific antibody can be recovered from the culture medium in the form of a secreted polypeptide, although it can also be recovered from the host cell lysate when produced directly without a secretory signal. If the bispecific antibody is bound to the membrane, it can be released from the membrane using a suitable detergent solution (eg, Triton-X 100).

After isolation, a bispecific antibody comprising two human heavy chains and a single human light chain derived from a rearranged gene sequence of human light chain variable region selected from the Vκ1-39Jκ5 and Vκ3-20Jκ1 sequences comprising a substitution of al minus a non-histidine codon with a histidine codon, is screened to determine its ability to exhibit pH-dependent binding to one, preferably both of its antigens. The ability of bispecific antibodies to bind their antigens differently to neutral and acidic pH of (for example, their ability to demonstrate a reduced 30 t1 / 2 at acidic pH compared to neutral pH) can be determined by a variety of techniques. available in the art and described in the following examples, for example, the BIACORE ™ assay.

Additional methods for the generation of pH-dependent antigen-binding proteins 35

Various methods of generating antigen-binding proteins with pH-dependent antigen-binding properties in genetically modified non-human animals described herein are described herein. Also described herein are methods for generating antigen-binding proteins with pH-dependent antigen-binding properties in vitro. Such methods may involve the generation of various components of the antigen binding proteins in vivo in genetically modified non-human animals and then the modification and reassembly thereof in vitro outside an organism in the form of protein complexes expressed in cultures. mammalian cell phones

In one aspect, the method of generating antigen-binding proteins with pH-dependent antigen-binding properties utilizes an antigen-binding protein sequence, for example, an antibody sequence, which is generated in a mouse comprising a limited repertoire of light chain variable region V and J segments, for example, human light chain variable region V and J segments, "universal light chain" or "common light chain" mouse ("ULC" mouse), such as the mouse described in the US Application Publications. No. 2011/0195454, 2012/0021409, 2012/0192300 and 2013/0045492. In one aspect, method 50 of generating antigen-binding proteins with pH-dependent antigen-binding properties utilizes an antigen-binding protein sequence that is generated in a mouse that comprises a single gene sequence of variable chain region. Light human rearranged. In one aspect, the method uses an antigen-binding protein generated in a mouse that comprises a single rearranged gene sequence of human light chain variable region selected from human Vκ1-39Jκ5 and human Vκ3-20Jκ1. 55

In one aspect, the method of generating an antigen-binding protein, for example, an antibody, with the pH-dependent antigen-binding properties comprises the selection of a first antibody that binds to an antigen of interest (e.g., binds to an antigen of interest with a desired affinity), the modification of an immunoglobulin light chain nucleotide sequence of the first antibody to comprise a substitution of at least one non-histidine codon with a histidine codon, the expression of an immunoglobulin heavy chain of the first antibody and the modified immunoglobulin light chain in a cell and the selection of a second antibody expressed in the cell that retains binding to the antigen of interest (for example, retains the desired affinity for the antigen of interest) at neutral pH and shows a reduced binding to the antigen of interest at an acidic pH. 65

In one aspect, the method of generating an antigen-binding protein, for example, an antibody, with pH-dependent antigen-binding properties comprises the selection of an immunoglobulin heavy chain of an antibody (e.g., obtained from an animal non-human, for example, a mouse, for example, a ULC mouse) comprising an immunoglobulin light chain having a single rearrangement of the human immunoglobulin light chain variable region in which the antibody binds to a 5-antigen interest (for example, binds to an antigen of interest with a desired affinity); the modification of the immunoglobulin light chain nucleic acid sequence so that the rearranged human immunoglobulin light chain variable region sequence only comprises a substitution of at least one non-histidine codon with a histidine codon; the expression of the selected immunoglobulin heavy chain and the immunoglobulin light chain comprising the replacement of at least one amino acid with a histidine in its variable domain; and selecting an antibody that retains the binding to the antigen of interest at a neutral pH (for example, that retains the desired affinity for the antigen of interest) while showing a reduced binding to the antigen of interest at an acidic pH. In various aspects, the immunoglobulin heavy chain derives from a rearrangement of human heavy chain variable gene segments (human segments V, D and J).

 fifteen

In one aspect, the method of generating an antigen-binding protein, for example, an antibody, with pH-dependent antigen-binding properties comprises (1) immunizing a non-human animal, for example, a mouse, comprising a single gene sequence reordered from human light chain variable region and a repertoire of non-rearranged human heavy chain variable gene segments (segments V, D and J) with an antigen of interest and allowing a mouse to mount an immune response to said antigen, ( 2) select in the non-human animal, for example, in the mouse, an antibody that binds to the antigen of interest with a desired affinity, (3) isolate from the non-human animal, for example, from the mouse, a nucleotide sequence of an immunoglobulin heavy chain of the antibody that binds to the antigen of interest with a desired affinity, (4) determining the nucleotide sequence of said heavy chain, (5) modifying a nucleotide sequence gone from an immunoglobulin light chain containing the unique human rearranged immunoglobulin 25 light chain variable region to comprise a substitution of at least one non-histidine codon with a histidine codon, (6) expressing the immunoglobulin heavy chain of the antibody that binds to the antigen of interest with a desired affinity and the immunoglobulin light chain comprising histidine modification in a cell and (7) determining whether the antibody expressed in the cell retains antigen binding at a neutral pH, showing at the same time a reduced binding at an acidic pH. In one aspect, the antibody expressed in cell 30 has a desired affinity for the desired antigen at neutral pH. In various aspects, the immunoglobulin heavy chain derives from a rearrangement of human heavy chain variable gene segments (human V, D and J segments).

In one aspect, the mouse comprising a single rearranged human light chain variable region gene sequence is a "universal light chain" or "common light chain" mouse described in, for example, US Application Publications. .UU. No. 2011/0195454, 2012/0021409, 2012/0192300 and 2013/0045492. In one aspect, the only rearranged gene sequence of human light chain variable region is selected from the human Vκ1-39Jκ5 sequence and the human Vκ3-20Jκ1 sequence.

 40

In one aspect, the antigen of interest is selected from a soluble antigen, a cell surface antigen (eg, a tumor antigen) and a cell surface receptor. In a specific aspect, the cell surface receptor is an immunoglobulin receptor. In a specific aspect, the immunoglobulin receptor is an Fc receptor.

 Four. Five

In one aspect, the desired affinity of an antibody for an antigen expressed as a dissociation constant (KD) at a neutral pH is less than 10-6 M, for example, less than 10-1 M, for example, less than 10 -9 M, for example, less than 10-10 M, for example, less than 10-11 M, for example, less than 10-12 M.

As explained above, ULC mice, in one aspect, comprise a single gene sequence 50 rearranged variable human immunoglobulin light chain and express antibodies in response to the antigen, in which the affinity of the antibodies to the antigen is mediated primarily at through the heavy chains of their antibodies. These mice comprise a repertoire of human heavy chain variable segments (V, D and J), which are rearranged to encode a human heavy chain variable domain of an antibody that also comprises the light chain derived from the single rearranged chain variable sequence light human. 55 In one aspect, after exposure to the antigen, these mice use the diverse repertoire of human heavy chain variable segments (V, D and J) to generate an antibody with affinity and specificity for the antigen. Therefore, upon exposure to the antigen, the nucleotide sequence of an immunoglobulin heavy chain of the antibody generated in the ULC mice can be isolated and used to generate a desired binding protein that also comprises an immunoglobulin light chain derived from the single sequence. Human immunoglobulin light chain variable region 60 rearranged (for example, the only human immunoglobulin light chain variable region rearranged sequence with a substitution of at least one non-histidine codon with a histidine codon).

In one aspect of ULC mice, 90-100% of non-rearranged non-human VH gene segments are replaced with at least one non-rearranged human VH gene segment. In a specific aspect, all or substantially all (for example, 90-100%) of the V gene segments

Once the heavy chain of the antibody that binds to the antigen of interest is determined with the desired affinity, the heavy chain nucleotide sequence is isolated and sequenced. The sequence is cloned into a vector for expression in suitable host cells, for example, eukaryotic cells, for example, CHO cells. In one aspect, the sequence of a human heavy chain constant region is cloned downstream of the human heavy chain variable region sequence isolated from the mouse (eg, from the ULC mouse).

In one aspect, the method of generating an antigen binding protein with pH dependent antigen binding properties comprises modifying an immunoglobulin light chain nucleotide sequence, in particular the sequence of the single light chain variable region of rearranged human immunoglobulin, to comprise a replacement of at least one histidine no codon with a histidine codon. Various techniques for the modification of a nucleotide sequence are known in the art, for example, site-directed mutagenesis. In addition, a nucleotide sequence comprising the desired histidine substitution can be synthesized de novo. 25

In one aspect, the replacement of at least one non-histidine codon with a histidine codon comprises a substitution that results in the expression of one, two, three, four or more histidine residues. In one aspect, substitution or substitutions results in the expression of three or four histidine residues. In one aspect, the substitution or substitutions is in the light chain variable region of immunoglobulin. In one aspect, the substitution or substitutions is in the CDR codon, for example, in the CDR1, CDR3 and / or CDR3 codon. In one aspect, the substitution or substitutions is in the codon CDR3.

In one embodiment, in which the immunoglobulin light chain nucleic acid sequence comprises the Vκ1-39Jκ5 gene sequence, and the substitution or substitutions is in the CDR3 codon, the substitution results in the expression of a histidine in a position selected from 105, 106, 108, 111 and combinations thereof. In one embodiment, the substitutions result in the expression of histidines at positions 105, 106, 108 and 111. In one embodiment, the substitutions result in the expression of histidines at positions 105 and 106. In one embodiment, the substitutions result in the expression of histidines at positions 105 and 180. In one embodiment, the substitutions result in the expression of histidines at positions 105 and 111. In one embodiment, the substitutions result in the expression of histidines in the positions 106 and 108. In one embodiment, the substitutions result in the expression of histidines in positions 106 and 111. In one embodiment, the substitutions result in the expression of histidines in positions 108 and 111. In one embodiment, the substitutions result in histidine expression at positions 105, 106 and 108. In one embodiment, substitutions result in expiration. histidine sion at positions 105, 106 and 111. In one embodiment, the substitutions result in the expression of histidines at positions 105, 108 and 111. In one embodiment, the substitutions result in the expression of histidines in the positions 106, 108 and 111. Nucleic acid and amino acid sequences of CDR3 regions Vκ1-39Jκ5 comprising various histidine substitutions are represented in FIG. 2 and are included in the sequence listing.

 fifty

In one embodiment, in which the immunoglobulin light chain nucleic acid sequence comprises the Vκ3-20Jκ1 gene sequence, and the substitution or substitutions is in the CDR3 codon, the substitution results in the expression of a histidine in a selected position. between 105, 106, 107, 109 and combinations thereof. In one embodiment, the substitutions result in the expression of histidines at positions 105, 106, 107 and 109. In one embodiment, the substitutions result in the expression of histidines at positions 105 and 106. In one embodiment, the substitutions result in the expression of histidines at positions 105 and 107. In one embodiment, the substitutions result in the expression of histidines at positions 105 and 109. In one embodiment, the substitutions result in the expression of histidines in the positions 106 and 107. In one embodiment, the substitutions result in the expression of histidines in positions 106 and 109. In one embodiment, the substitutions result in the expression of histidines in positions 107 and 109. In one embodiment, the substitutions result in the expression of histidines at positions 105, 106 and 107. In one embodiment, the substitutions result in the former histidine pressure at positions 105, 106 and 109. In one embodiment, the substitutions result in the expression of histidines at positions 105, 107 and 109. In one embodiment, the substitutions result in the expression of histidines at positions 106 , 107 and 109. Nucleic acid and amino acid sequences selected from CDR3 Vκ3-20Jκ1 regions comprising 65 various histidine substitutions are depicted in FIG. 12 and are included in the sequence listing.

Once the immunoglobulin light chain sequence, for example, the human immunoglobulin light chain variable domain is modified to include histidine residues at the desired positions, the light chain nucleotide sequence is cloned into a vector for expression in suitable host cells, for example, eukaryotic cells, for example, CHO cells. In one aspect, the sequence of a human light chain constant region is cloned downstream of the modified nucleotide sequence of the human variable region.

In one aspect, vectors comprising a nucleotide sequence encoding the modified human immunoglobulin light chain and the selected human immunoglobulin heavy chain are coexpressed in a suitable host cell, for example, a eukaryotic host cell, for example, CHO cells , 10 to generate an antigen binding protein. Various host cells that can be used for expression are known in the art and are mentioned throughout the present specification.

An antigen-binding protein, for example, an antibody, generated in the host cell can be secreted in the cell supernatant, which is analyzed for proper expression and determine affinity for the original antigen at neutral pH. The antigen-binding protein can also be recovered from the cell lysate, or, if bound to the membrane, released from the membrane using a suitable detergent (eg, Triton-X). The antigen binding protein with the desired characteristics can be purified.

In one aspect, the antigen-binding protein comprising a modification or modifications with histidine 20 retains affinity for the antigen that is comparable to the affinity for the antigen of the same (original) antigen-binding protein that does not comprise a modification. or modifications with histidine. In one aspect, the affinity of the histidine modified antigen binding protein for the antigen of interest expressed as a dissociation constant (KD) at a neutral pH is less than 10-6 M, for example, less than 10-8 M, for example, less than 10-9 M, for example, less than 10-10 M, for example, less than 10-11 M, for example, less than 10-12 M. 25

In one aspect, the antigen binding protein, for example, an antibody, comprising histidine modifications described herein has pH dependent antigen binding properties. In one embodiment, the antigen binding protein comprising histidine modifications possesses enhanced pH dependent properties greater than an equivalent antigen binding protein without histidine modifications (antigen binding protein of the same amino acid sequence except for histidine modifications). In one aspect, the antigen binding protein described herein retains antigen binding at neutral pH (e.g., retains desired affinity for antigen at neutral pH), while exhibiting reduced binding at acidic pH. . In one aspect, the antigen binding protein, for example, the antibody, which is described herein, does not exhibit any antigen binding at acidic pH, while retaining antigen binding at neutral pH. In one aspect, an antigen-binding protein described herein has a decrease in the dissociative half-life (t1 / 2) at an acidic pH, compared to the dissociative half-life (t1 / 2) of the protein of antigen binding at a neutral pH, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times , at least about 25 times or at least about 30 times. In one aspect, an antigen binding protein described herein has a t1 / 2 at an acidic pH and 37 ° C of about 2 min or less. In one embodiment, an antigen binding protein described herein has a t1 / 2 at an acidic pH and 37 ° C of less than about 1 min. In one aspect, an antigen binding protein described herein has a t1 / 2 at an acidic pH and 25 ° C of about 2 min or less. In one aspect, an antigen binding protein described herein has a t1 / 2 at an acidic pH and 25 ° C of less than about 1 min.

In one aspect, the antigen-binding protein, for example, the antibody, comprising histidine modifications described herein, has an increased serum half-life after administration of a therapeutic dose to a subject compared to a half-life in serum after administration of an equivalent therapeutic dose of antigen-binding protein that does not comprise histidine modifications (eg, the original antigen-binding protein that does not comprise histidine modifications). In some aspects, the increase in serum half-life after administration of a dose of the antigen-binding protein comprising histidine modifications described herein during a half-life in serum after administration of the same dose of the antigen binding protein that does not comprise histidine modifications is about 2 times, for example, about 5 times, for example, about 10 times, for example, about 15 times, for example, about 20 times or more. In one aspect, the serum half-life is at least about 1 day, for example, at least about 2 days, for example, at least about 7 60 days, for example, at least about 14 days, for example, at least about 30 days, for example, at least about 60 days.

In addition to the in vitro methods for the generation of antigen-binding proteins with pH dependent antigen-binding properties described above, it is also described herein that 65 antigen-binding proteins, for example, antibodies, generated by said method. Furthermore, said method can be used to generate multi-specific antibody binding protein, for example, bispecific, by selecting two different human immunoglobulin heavy chains that bind to a common (universal) light chain in a mouse, determining of the nucleotide sequences of the heavy chains, the universal light chain modification to comprise histidine substitutions as described above and the coexpression of two human heavy chains with a single modified universal light chain 5 with histidine in a host cell. Various steps for the generation of an antigen binding protein described above may be applicable to the method of generating a bispecific antigen binding protein. The bispecific antigen binding protein, which is confirmed to have the affinity for the desired antigen or antigens and the pH dependent antigen binding properties, can be purified. Thus, bispecific antibodies comprising two human heavy chains and a single human light chain comprising a human light chain variable domain sequence encoded by a human variable region gene are described, for example, the gene of Vκ1-39Jκ5 or Vκ3-20Jκ1 human variable region comprising a substitution of at least one non-histidine codon with a histidine codon.

Also described herein are constructions used in the production of an antigen-binding protein comprising human immunoglobulin heavy chain and human immunoglobulin light chain comprising histidine substitutions. Also described herein are host cells that express antigen-binding proteins, for example, antibodies, which are described herein.

 twenty

Examples

The following examples are provided in order to describe to those of ordinary skill in the art how to make and use the methods and compositions described, and are not intended to limit the scope of what the inventors consider their disclosure. Efforts have been made to ensure accuracy with respect to the numbers used (eg, quantities, temperature, etc.) but some experimental errors and deviations must be taken into account. The Examples do not include detailed descriptions of conventional methods that are well known to those of ordinary skill in the art (molecular cloning techniques, etc.). Unless otherwise indicated, the parts are parts by weight, the molecular weight is average molecular weight, the temperature is indicated in degrees Celsius and the pressure is atmospheric or close to it. 30

Example 1. Identification of histidine residues in antigen-specific human light chains

The generation of a common light chain mouse (eg, common light chain mouse Vκ1-39 or Vκ3-20) and antigen specific antibodies in those mice is described in US Pat. No. 35 13 / 022,759, 13 / 093,156 and 13 / 412,936 (Publications No. 2011/0195454, 2012/0021409 and 2012/0192300, respectively). Briefly, a light chain direction vector of the rearranged human germ line was made using VELOCIGENE® technology (see, for example, US Patent No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21 (6): 652-659) to modify clones of mouse genomic bacterial chromosome (BAC) genetically engineered genomic constructs to contain a single light chain region of human germline line rearranged and inserted into an endogenous κ light chain locus that had previously been modified to suppress endogenous κ variable and gene binding segments. Then, directed BAC DNA was used to electroporate mouse ES cells to create modified ES cells to generate chimeric mice expressing a Vκ1-39Jκ5 or Vκ3-45 20Jκ1 region of rearranged human germ lineage. Targeted ES cells were used as donor ES cells and were introduced into an 8 cell stage mouse embryo by the VELOCIMOUSE® method (see, for example, US Patent No. 7,294,754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses Nature Biotech. 25 (1): 91-99). VELOCIMICE® were identified that independently carried a light chain region Vκ1-39Jκ5 or 50 Vκ3-20Jκ1 of genetically modified human germ line using genotyping using an allele modification assay (Valenzuela et al., Cited above) that detects the presence of the only light chain region of rearranged human germ line.

Mice carrying a genetically engineered modified human germline light chain locus (ULC mice) were reproduced with mice containing a replacement of the endogenous mouse heavy chain variable gene locus with the heavy chain variable gene locus human (see US 6,596,541; the VELOCIMMUNE® mouse, Regeneron Pharmaceuticals, Inc.).

The VELOCIMMUNE® mouse containing a single light chain region of rearranged human germ line 60 was exposed to an antigen of interest and antibodies comprising a universal light chain (eg, Vκ1-39Jκ5) were isolated and sequenced. The amino acid sequences of selected light chains (A-K) of antigen specific human antibodies generated in a common Vκ1-39Jκ5 light chain mouse were aligned. Histidine mutations were identified in light chain CDRs derived from Vκ1-39 for a selected number of human antigen-specific antibodies (FIG. 1). The partial amino acid sequence 65 of the variable domain Vκ1-39Jκ5 of germ line is shown above the alignments and is set forth in SEQ ID NO: 1, the amino acid sequence of the complete variable domain is set forth in SEQ ID NO: 80.

Example 2. Modification by genetic engineering and characterization of human universal light chain antibodies substituted with histidine

 5

Example 2.1 Genetic engineering modification of histidine residues in a human rearranged light chain of germ line

Histidine residues were introduced genetically engineered into a rearranged human Vκ1-39Jκ5 light chain using site-directed mutagenesis primers specifically designed to introduce genetically modified histidine residues at positions Q105, Q106, Y108 and P111 of the light chain Human Vκ1-39Jκ5. Site-directed mutagenesis was performed using molecular techniques known in the art (eg, QuikChange II XL Site Directed Mutagenesis Kit, Agilent Technologies). The locations of genetically engineered residues in the CDR3 are shown in FIG. 2, the histidine substituted CDR3 nucleic acid sequences depicted in FIG. 2 are set forth in SEQ ID NO: 4, 6, 8, 10, 12, 15 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32 (the corresponding amino acid sequences are set out in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and 33). The nucleic acid and amino acid sequences of the rearranged Vκ1-39Jκ5 CDR3 germline strain are set forth in SEQ ID NO: 2 and 3, respectively.

Example 2.2 Construction and expression of light chains modified by genetic engineering with histidine 20

Light chains derived from human Vκ1-39 containing histidine residues introduced by genetic engineering of germ line made according to Example 2 were constructed and paired with various human heavy chains (labeled 1-5), specific for a surface receptor human cell, to analyze the expression in CHO cells. The five specific human heavy chains for a human cell surface receptor that were paired with histidine-substituted Vκ1-39 derived light chains were obtained from mice that had a single rearranged human light chain (a rearranged light chain Vκ1-39 / Jκ5 human; see US2011 / 0195454A1).

Enzyme-linked immunosorbent assay (ELISA): Antibody secretion from CHO 30 cells was detected using an Fc ELISA, for light chains with histidine modifications indicated with five different heavy chains. Light and heavy chain sequences (except modifications) were generated in mice that had a single rearranged human light chain (eg, a human Vκ1-39 / Jκ5 rearranged light chain; see US2011 / 0195454A1). The capture antibody was goat anti-human IgG and the detection antibody was goat anti-human HRP (Fc gamma specific). The results are shown in FIG. 3. Heavy ULC +: specific heavy chain and light chain derived from unmodified human Vκ1-39. As shown in FIG. 3, expression was detected in almost all mutants.

Immunoblotting of proteins. Expression in CHO cell supernatants of antigen specific heavy chains paired with genetically modified light chains with histidine was further analyzed by Western blotting. The samples were grown on a 4-12% tris gel-glycine. The results using a selected heavy chain (heavy chain 3) are shown in FIG. 4. ULC refers to a light chain derived from rearranged human Vκ1-39 (as described above).

Example 2.3 Determination of binding affinity of light chains modified by genetic engineering with histidine

Equilibrium dissociation constants (KD), dissociative half-lives (t1 / 2) and other kinetic parameters were determined for antibody supernatants selected by SPR (surface plasmon resonance) using a BIACORE ™ T200 instrument (GE Healthcare). The kinetics was measured at pH 7.4 and at pH 5.75. The results 50 are shown in FIG. 5E - 5A.

Numerical values were obtained for the kinetic binding properties (for example, ka, kd, KD, t1 / 2, etc.) of antibodies that bind to immunogen at neutral pH (pH 7.4) and at acidic pH (pH 5, 75) using a real-time surface plasmon resonance biosensor (Biacore T200). A Biacore CM5 sensor chip was derivatized with a human anti-Fc mouse antibody to capture antibodies from the supernatant. Then, a single concentration (50 nM) of immunogen was injected onto the surface captured by antibody at a flow rate of 30 µl / min. The antibody-antigen association was monitored for 2.5 minutes and then antigen dissociation of the captured antibody was monitored for 8 minutes. The kinetic association (ka) and dissociation (kd) rate constants were determined by processing and adjusting the data to a 1: 1 junction with a 60 mass transport model using the Biacore T200 Evaluation version 1.0 software. Equilibrium dissociation constants (KD) and dissociative half-lives (t1 / 2) were calculated from kinetic velocity constants such as: KD (M) = kd / ka; and t1 / 2 (min) = (In2 / (60 * kd).

As shown in FIG. 5, in an antibody binding assay to a cell surface receptor, two out of five 65 histidine modified common light chain antibodies (VRD1-39 / Jκ5 modified light chain histidine) that matched human heavy chains antigen-specific, presented antigen binding (eg, to a cell surface receptor) with different affinities at pH 7.4 and pH 5.75. Antibodies with histidine modifications that retain binding at pH 7.4, but have a low or no detectable binding at pH 5.75 are desirable. Histidine-modified antibodies that have a t are desirable.

The antigen binding data for three antibodies comprising common histidine modified light chains and three antigen specific heavy chains (labeled 2, 3 and 6) at different pHs are further summarized in FIG. 6. These antibodies showed a significant drop in antigen binding at pH 5.75 compared to pH 7.4, as demonstrated, for example, by reduction in t1 / 2 or non-binding detected at pH. 5.75.

Example 3. Modification by genetic engineering and characterization of a genetically modified mouse comprising a universal light chain Vκ1-39Jκ5 substituted with histidine

 fifteen

Example 3.1 Construction of direction vector for histidine residues introduced by genetic engineering in a variable region chain of rearranged human light chain

A transgenic mouse is produced that contains a rearranged human light chain gene having histidine residues introduced by genetic engineering into a CDR region of the human light chain using direction vectors 20 made by conventional molecular cloning techniques known in the art.

Briefly, various reordered human germline light chain direction vectors are produced using VELOCIGENE® technology (see, for example, US Patent No. 6,586,251 and Valenzuela et al. (2003) High -throughput engineering of the mouse genome coupled with high-resolution expression analysis, 25 Nature Biotech. 21 (6): 652-659) to modify the bacterial artificial chromosome DNA (BAC) mouse genomics so that It contains a single light chain region of rearranged human germ line and is inserted into an endogenous κ light chain locus that was previously modified to suppress the endogenous κ variable and bind the gene segments. The light chain region of rearranged human germ line is modified at one or more nucleotide positions within the light chain sequence to encode histidine residues that are not normally present at the respective locations of the germ line sequence. Direction vectors are electroporated into mouse embryonic stem cells (ES) and confirmed using a quantitative PCR assay (eg, TAQMAN ™).

Specifically, a strategy for the construction of these direction vectors is shown in FIG. 8A - 8F. A plasmid used to generate an address vector for the common (universal) light chain mouse ("ULC mouse", described in, for example, US2011 / 0195454A1), containing pBS + FRT-Ub-Hyg-FRT + mouse leader Vκ3-7 + human Vκ1-39Jκ5 was modified by site-directed mutagenesis (QuickChange II XL Kit) to replace Q105, Q106, Y108 and P111 or P106, Y108 and P111 with histidine residues in the CDR3 region using primers of site-directed mutagenesis shown in FIG. 7 (see FIG. 8A for this stage of genetic engineering modification). The resulting vectors (H105 / 106/108/111 and H106 / 108/111) were further modified and ligated into a vector comprising mouse Igκ constant region, mouse enhancers, a 3 'mouse homology arm and a cassette SPEC (FIG. 8B). The additional modification involved ligation in a vector bearing the 5 'mouse arm and comprising Frt-Ub-NEO-Frt cassette (FIG. 8B). The resulting direction vectors were electroporated into ES cells comprising deletion of the mouse Igκ variable locus (which comprised variable κ and linked gene segments) (FIG. 8C-8F).

Positive ES cell clones were confirmed by the use of a modification of the allele assay (Valenzuela et al.) Using specific probes for the modified Vκ1-39Jκ5 light chain region by genetic engineering inserted into the endogenous κ light chain locus. Primers and probes used in the assay are shown in Table 1 below and are set forth in the Sequence Listing; probe locations are represented in FIG. 8C-8F.

Table 1: Primers and probes used for ES cell scanning

 Probe name

 Test Probe Sequence Primer 5 'Primer 3'

 Neo

 GOA TGGGCACAACA GACAATCGGCTG (SEQ ID NO: 38) GGTGGAGAGG CTATTCGGC (SEQ ID NO: 39) GAACACGGCGG CATCAG (SEQ ID NO: 40)

 ULC-m1

 GOA CCATTATGATGC TCCATGCCTCTC AGGTGAGGGT ACAGATAAGTG TGACAAATGCCC TAATTATAGTGAT

 TGTTC (SEQ ID NO: 41) TTATGAG (SEQ ID NO: 42) CA (SEQ ID NO: 43)

 Probe name

 Test Probe Sequence Primer 5 'Primer 3'

 1633h2 (specific for Vκ1-39Jκ5)

 GOA ATCAGCAGAAAC CAGGGAAAGCCC CT (SEQ ID NO: 44) GGGCAAGTCA GAGCATTAGCA (SEQ ID NO: 45) TGCAAACTGGAT GCAGCATAG (SEQ ID NO: 46)

 mlgKd2

 Retention GGCCACATTCCA TGGGTTC (SEQ ID NO: 47) GCAAACAAAAA CCACTGGCC (SEQ ID NO: 48) CTGTTCCTCTAAA ACTGGACTCCAC AGTAAATGGAAA (SEQ ID NO: 49)

 mlgKp15

 Retention GGGCACTGGATA CGATGTATGG (SEQ ID NQ: 50) CACAGCTTGTG CAGCCTCC (SEQ ID NO: 51) AGAAGAAGCCTG TACTACAGCATCC GTTTTACAGTCA (SEQ ID NO: 52)

The NEO selection cassette introduced by address constructs was suppressed by transfection of ES cells with a plasmid expressing FLP (FIG. 8C and 8E). Optionally, the neomycin cassette can be removed by reproducing mice expressing FLP recombinase (eg, US 6,774,279). Optionally, the neomycin cassette is preserved in the mice. 5

The targeted ES cells described above were used as donor ES cells and were introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, for example, US Patent No. 7,294,754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses Nature Biotech. 25 (1): 91-99. 10 VELOCIMICE® were produced that independently carried a gene genetically engineered human light chain containing histidine residues mutated in one or more positions along the sequence, from targeted ES cells described above.

Pups were genotyped and heterozygous offspring were selected for the human light chain modified with histidine 15 by genetic engineering to characterize the expression of the light chain and the binding capabilities of the expressed antibodies. Primers and probes are listed for genotyping of mice that specifically comprise a universal light chain gene with either three (H106 / 108/111; "1930") or four (H105 / 105/108/111; "1927") modifications with histidine in Table 2 below and are set out in the Sequence Listing. Mice containing histidine modification in their universal light chains are referred to herein as "HULC" mice (universal light chain mice with histidine).

Table 2: Primers and probes used for genotyping

 Probe name

 Test Probe Sequence Primer 5 'Primer 3'

 1927jxn3

 GOA 1927 (4 His) mouse-specific ACCATAGTCACA GTACCCA (SEQ ID NO: 53) AGCAGTCTGCA ACCTGAAGATTT (SEQ ID NO: 54) CCCTTGGCCGAA GGTGAT (SEQ ID NO: 55)

 1930jxn3

 GOA 1930 (3 His) mouse specific ATAGTCACAGTA CCCATCC (SEQ ID NO: 56) AGTCTGCAACC TGAAGATTTTGC (SEQ ID NO: 57) CCCTTGGCCGAA GGTGAT (SEQ ID NO: 58)

Example 3.2. Analysis of the immune response to antigen in mice with universal light chains substituted with histidine

A cell surface receptor ("A antigen") was used as the immunogen to immunize mice that were heterozygous for the expression of a pre-ordered human kappa light chain using Vκ1-39 and Jκ5 which has 4 histidine substitutions in CDR3 (in hereinafter "HULC 1927") or heterozygous for the expression of a pre-ordered human kappa light chain using Vκ1-39 and Jκ5 having 3 histidine substitutions in CDR3 (hereinafter "HULC1930 ") or homozygous TS mice. Preimmune serum was collected from the mice before the start of immunization. The immunogen was administered at 2.35 µg of protein for initial sensitization immunization mixed with 10 µg of CpG oligonucleotide as an adjuvant (Invivogen) in a volume of 25 µl through the plantar pad (a.p.). Subsequently, the mice were reinforced through the same route with 2.35 µg of antigen A together with 10 µg of CpG and 25 µg of Adju-Phos (Brenntag) as adjuvants on days 3, 6, 11, 13, 17 , 20 for a total of 6 reinforcements. The mice bled on days 15 and 22 after the 4th and 6th impulse, respectively. Its antiserum was tested for antibody titers for Antigen A.

 40

Serum titres of antibodies against immunogen were determined by a conventional ELISA. To perform the ELISA, 96-well microtiter plates (Thermo Scientific) were coated at 2 µg / ml with Antigen A in phosphate buffered saline (PBS, Irvine Scientific) overnight at 4 ° C. The next day, the plates were washed with phosphate buffered saline containing 0.05% Tween 20 (PBS-T, Sigma-Aldrich) four times using a plate washer (Molecular Devices). The plates were then blocked with 250 µl of 0.5% bovine serum albumin (BSA, Sigma-Aldrich) in PBS and incubated for 1 hour at room temperature. Then, the plates were washed four times with PBS-T. Serums from immunized mice and pre-immune sera were serially diluted three times in 0.5% BSA-PBS starting at 1: 300 or 1: 1000, 5 blocks were added in duplicate and then incubated for 1 hour. at room temperature. The last two wells are left blank for use as a secondary antibody control (background control). Then, the plates were washed again four times with PBS-T in a plate washer. Then goat anti-mouse IgG-Fc-horseradish peroxidase (HRP) conjugated secondary antibody (Jackson Immunoresearch) was added to the plates at a dilution of 1: 5000/1: 10000 and incubated for 1 hour at room temperature. Then, the plates were washed eight times with PBST and developed using TMB / H

 fifteen

The immune response induced in mice against the injected immunogen is represented as antibody titers, which is defined as the reciprocal of the highest serum dilution at which the antigen binding absorbance is twice higher than the background level. Therefore, the higher the number, the greater the humoral immune response against the immunogen. Immunogen induced antibody titres were very high in both strains of HULC mice and in TS mice, with no significant differences observed among the 20 strains (FIG. 9).

Example 3.3. Generation of pH sensitive monoclonal antibodies

When a desired immune response against the immunogen was achieved in both strains of HULC mice and 25 in TS mice, splenocytes were collected from each mouse strain and fused with mouse myeloma cells to generate hybridoma cells, which were allowed to grow. in 96-well plates. After 10 days of growth, supernatants from each well containing hybridoma cells were screened by immunogen specific ELISA to identify positive antigen binding samples. For the ELISA, 96-well micro-titration plates were coated with 1 ug / ml of an anti-myc polyclonal antibody (Novus Biologicals, No. 30 NB600-34) overnight at 4 ° C to immobilize the antigen labeled with myc, followed by blocking with a 0.5% (w / v) BSA solution in PBS. The plates were washed, the antigen solutions were added to the plates at a concentration of 1 µg / ml and allowed to bind to the coated plate for 1 hour at room temperature. Subsequently, hybridoma cell supernatants were added to the wells at a 1:50 dilution and allowed to bind for 1 hour at room temperature. Antibodies bound to the plate were detected using a polyclonal anti-mouse IgG antibody conjugated to HRP (Jackson Immunoresearch, No. 115-035-164). TMB substrates were added to the plates (BD Biosciences, No. 51-2606KC / 51-2607KC) and colorimetric signals were developed according to the protocol recommended by the manufacturer. The absorbance was recorded at 450 nm in a Victor Wallac plate reader. Antigen positive samples defined as having an OD equal to or greater than 0.5 (having an OD of approximately 0.1 at baseline) were subjected to affinity scanning 40 using a real-time surface plasmon resonance biosensor ( Biacore 4000).

Kinetic binding parameters (eg, ka, kd, KD, t1 / 2, etc.) were recorded for binding of the antibody to the immunogen at neutral pH (pH 7.4) and at acidic pH (pH 6.0). A Biacore CM4 sensor chip was derivatized with a polyclonal goat anti-mouse Fc antibody to capture antibodies from the supernatant. Then, a single concentration (100 nM) of immunogen was injected onto the surface captured by antibody at a flow rate of 30 µl / min. The antibody-antigen association was monitored for 1.5 minutes and then the antigen cleavage of the captured antibody was monitored for 2.5 minutes. The kinetic constants of association speed (ka) and dissociation (kd) were determined by processing and adjusting data to a 1: 1 junction with a mass transport model using the Biacore 4000 Evaluation version 1.0 software. 50 equilibrium dissociation constants (KD) and dissociative half-lives (t1 / 2) were calculated from the kinetic velocity constants such as: KD (M) = kd / ka; and t1 / 2 (min) = In2 / (60 * kd). A set of samples that showed a decrease in binding to pH 6.0 compared to that of pH 7.4 (sensitive pH) as well as a set of control samples that showed no significant velocity changes between pH 7.4 and pH 6.0 (pH insensitive controls) were selected to be produced by cloning. FIG. 10 represents the comparison of the number of total antigen positive 55 and the number of antigen positive showing a pH sensitive antigen binding for HULC and TS mice.

Among the antigen-positive ones, 18 and 7 isolated clones of two heterozygous HULC1927 and two HULC1930 mice respectively, and 1 TS mouse clone were produced monoclonally. The supernatants of the 60 monoclonal hybridomas were subjected to dissociation rate (dissociation rate) analysis of the antigen at neutral and low pH and cellular sediments were used for sequencing of light chain variable domain DNA.

65 Example 3.4. Sequencing and somatic hypermutations in the CDR3 region of universal light chain mice with histidine based on Vκ1-39Jκ5

Cellular sediments of monoclonal hybridomas of HULC and TS mice were used for DNA sequencing of the light chain variable domain. Of the 26 clones made in a monoclonal manner (see Example 3.3 above) 5 and sequenced, it was confirmed that 15 used a HULC or TS mouse light chain (MM and NN, see Table 4). 14 clones were derived from HULC heterozygous mice (mice 1927 or 1930) and 1 derived from a TS mouse (OO, see Table 4).

Of the 14 antigen-positive samples derived from HULC heterozygous mice, 12 of the 10 monoclonal antibodies used their corresponding HULC light chain, while 2 used a TS mouse light chain. All but one of the antibodies that used HULC retained all of the histidine mutations introduced as shown in Table 3 (italicized antibody). Sequencing of clone AA produced 2 different HULC sequences, which is reflected by two entries in Table 3.

 fifteen

Table 3: Number of histidine insertions and somatic hypermutations conserved in light chain sequences of clones using the HULC light chain

 Light chain sequences of mice using HULC

 Clone name

 Mouse strain No. of His mutations conserved in CDR3 No. of somatic hypermutations in the conserved framework region No. of somatic hypermutations in CDR

 AA (Sequence 1)

 1927 4 3 0

 AA (Sequence 2)

 1927 4 1 1

 BB

 1927 4 3 3

 DC

 1927 4 0 0

 DD

 1927 3 1 1

 EE

 1927 4 2 2

 FF

 1927 4 0 1

 GG

 1927 4 1 1

 H H

 1927 4 2 0

 II

 1930 3 1 1

 JJ

 1930 3 4 5

 Kk

 1930 3 1 2

 LL

 1930 3 1 0

Example 3.5 PH-dependent binding of monoclonal antibodies generated in universal light chain mice with histidine based on Vκ1-39Jκ5 20

In order to further evaluate the pH-dependent binding characteristics of monoclonal antibodies isolated from HULC and TS mice, binding experiments were performed in which the antibody / antigen association phase was observed at neutral pH and the antibody dissociation phase. / antigen was observed at either neutral or acidic pH. 25

A Biacore CM4 sensor chip was derivatized with a polyclonal mouse anti-Fc rabbit antibody. Monoclonal antibody supernatants were captured on the surface of mouse anti-Fc sensor. Two concentrations of the immunogen, 50 nM (in duplicate) and 16.7 nM, were injected onto the surface captured with monoclonal antibody at a flow rate of 30 µl / min. The antigen-antibody association was monitored at pH 7.4 for 4 minutes and then the antigen dissociation of the captured monoclonal antibody was monitored for 15 minutes at pH 7.4 or 6.0. Dissociation rate constants (kd) were determined by data processing and adjustment using the Scrubber version 2.0 curve adjustment software and are shown in Table 4. Dissociative half-lives (t1 / 2) were calculated from the velocity constants of dissociation such as: t1 / 2 (min) = (In2 / kd) / 60 and are shown in Table 4. Sensograms are shown graphically representing the association / dissociation characteristics of several antibodies listed in Table 4 under various conditions pH in FIG. 11. The individual lines in each graph represent binding responses at different concentrations of the respective antibodies. All experiments were performed at 25 ° C. The values of dissociative half-life (t1 / 2) are indicated above the respective sensograms. The response is measured in UR.

40

Example 4. Genetic engineering modification of a genetically modified mouse comprising a universal light chain Vκ3-20Jκ1 substituted with histidine

A mouse comprising a common Vκ3-20Jκ1 light chain was generated as described in, for example, US Pat. No. 13 / 022,759, 13 / 093,156, 13 / 412,936 and 13 / 488,628 (Publications No. 5 2011/0195454, 2012/0021409, 2012/0192300 and 2013/0045492, respectively) and in Example 1 above. The amino acid sequence of the universal light chain variable domain Vκ3-20Jκ1 of germ line is set forth in SEQ ID NO: 59.

Histidine substitutions were introduced into the universal light chain direction vector Vκ3-20Jκ1 and mice 10 generated therefrom using a strategy similar to that described above in Example 3 for histidine modified universal light chain mice Vκ1-39Jκ5 ( HULC 1927 and 1930).

Briefly, the strategy to generate a histidine modified Vκ3-20Jκ1 universal light chain direction vector is summarized in FIG. 14A-14D. A plasmid used to generate a common (universal) light chain mouse direction vector ("ULC mouse", is described in, for example, US2011 / 0195454A1), which contained pBS + FRT-Ub-Hyg-FRT + mouse leader Vκ3-7 + human Vκ3-20Jκ1 was modified by site-directed mutagenesis (QuickChange Lightning Kit) to replace Q105, Q106, Y107 and S109 or Q105, Q106 and S109 (see alignment in FIG. 12) with histidine residues in the CDR3 region using site-directed mutagenesis primers shown in FIG. 13 (see FIG. 14A for this stage of modification by genetic engineering). The resulting vectors (H105 / 106/107/109 and H105 / 106/109) were further modified and ligated into a vector comprising mouse Igκ constant region, mouse enhancers, a 3 'mouse homology arm and a cassette SPEC (FIG. 14B). The additional modification involved ligation in a vector bearing a 5 'mouse arm and comprising Frt-UB-NEO-Frt cassette (FIG. 14B). The resulting direction vectors were electroporated into ES cells comprising suppression of the mouse Igκ variable locus (which comprises variable and κ binding gene segments) (FIG. 14C-14D).

Positive ES cell clones were confirmed by the use of an allele assay modification (Valenzuela et al.) Using specific probes for the Vκ3-20κJ1 light chain region modified by genetic engineering inserted into the endogenous κ light chain locus. Primers and probes used in the assay are shown in Table 5 below and are set forth in the Sequence Listing; probe locations are represented in FIG. 14C-14D.

Table 5: Primers and probes used to explore ES cells

 Probe name

 Test Probe Sequence Primer 5 'Primer 3'

 Neo

 GOA TGGGCACAACA GACAATCGGCTG (SEQ ID NO: 38) GGTGGAGAGG CTATTCGGC (SEQ ID NO: 39) GAACACGGCGG CATCAG (SEQ ID NO: 40)

 ULC-m1

 GOA CCATTATGATGCT CCATGCCTCTCT GTTC (SEQ ID NO: 41) AGGTGAGGGT ACAGATAAGTG TTATGAG (SEQ ID NO: 42) TGACAAATGCCC TAATTATAGTGAT CA (SEQ ID NO: 43)

 1635h2 (specific for Vκ3-20Jκ1)

 GOA AAAGAGCCACCC TCTCCTGCAGGG (SEQ ID NO: 65) TCCAGGCACCC TGTCTTTG (SEQ ID NO: 66) AAGTAGCTGCTG CTAACACTCTGAC T (SEQ ID NO: 67)

 mlgKd2

 Retention GGCCACATTCCA TGGGTTC (SEQ ID NO: 47) GCAAACAAAAA CCACTGGCC (SEQ ID NO: 48) CTGTTCCTCTAAA ACTGGACTCCAC AGTAAATGGAAA (SEQ ID NO: 49)

 mlgKp15

 Retention GGGCACTGGATA CGATGTATGG (SEQ ID NQ: 50) CACAGCTTGTG CAGCCTCC (SEQ ID NO: 51) AGAAGAAGCCTG TACTACAGCATCC GTTTTACAGTCA (SEQ ID NO: 52)

 35

The NEO selection cassette introduced by the target constructs is suppressed by transfection of ES cells with a plasmid that expresses FLP (FIG. 14C and 14 RE). Optionally, the neomycin cassette can be removed by reproducing mice expressing FLP recombinase (eg, US 6,774,279). Optionally, the neomycin cassette is preserved in the mice.

 40

The targeted ES cells described above were used as donor ES cells and were introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, for example, US Patent No. 7,294,754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses Nature Biotech. 25 (1): 91-99. VELOCIMICE® were produced that independently carried a gene of Genetically modified human light chain containing histidine residues mutated in one or more positions along the sequence, from targeted ES cells described above.

Pups were genotyped and heterozygous offspring were selected for the histidine modified human light chain by genetic engineering to characterize the expression of the light chain and the binding capabilities of the expressed antibodies. Primers and probes for genotyping of mice that specifically comprise a universal light chain gene with either three (H105 / 106/109; "6183") or four 10 (H105 / 105/108/111; "6181") are listed Histidine modifications in Table 6 below and are set forth in the Sequence Listing. Mice containing histidine modification in their universal light chains are referred to herein as "HULC" mice (universal light chain mice with histidine).

Table 6: Primers and probes used for genotyping 15

 Probe name

 Test Sequence of the probe Primer 5 'Primer 3'

 hVI494-1

 GOA 6181 (4 CTGTCATCACCA GCAGACTGGAG CCGAACGTCCAA

 His specific mouse TGG (SEQ ID NO: 68) CCTGAAGATTTT (SEQ ID NO: 69) GGTGAGTG (SEQ ID NO: 70)

 hVI495-1

 GOA 6183 (3 His) mouse-specific TACTGTCATCACT ATGG (SEQ ID NO: 71) GCAGACTGGAG CCTGAAGATTT (SEQ ID NO: 72) CCGAACGTCCAA GGTGAGTG (SEQ ID NO: 73)

Mice are immunized with antigen of interest and tested for the ability to generate antibodies with pH-dependent binding.

Example 5. Reproduction of mice comprising a human universal light chain mouse rearranged with histidine (HULC)

This Example describes several other genetically modified mouse strains that can be reproduced to any of the HULC mice described herein to create multiple genetically modified mouse strains that house multiple genetically modified immunoglobulin loci. 25

Inactivated gene (KO) of endogenous IgA. To optimize the use of the genetically modified light chain locus, any one of the HULC animals described above (for example, comprising universal light chain substituted with histidine Vκ1-39Jκ5 or Vκ3-20Jκ1) can be reproduced to another mouse containing a deletion in the endogenous λ light chain locus. In this way, the progeny obtained will express, as its only light chain, the light chain region of rearranged human germ line replaced with histidine as described in Examples 3 and 4 above. Reproduction is performed by conventional techniques recognized in the art and, alternatively, by a commercial breeder (for example, The Jackson Laboratory). Mouse strains carrying a light chain locus replaced with genetically modified histidine are screened and a deletion of the endogenous λ light chain locus is scanned to determine the presence of the single light chain region and the absence of λ light chains of endogenous mouse.

Humanized endogenous heavy chain locus. Mice carrying a genetically modified human germline strain chain locus (HULC mice) are reproduced with mice containing a replacement of the endogenous mouse heavy chain variable gene locus with the human heavy chain variable gene locus (see US 6,596,541; the VELOCIMMUNE® mouse, Regeneron Pharmaceuticals, Inc.). The VELOCIMMUNE® mouse comprises a genome comprising variable human heavy chain regions operatively linked to endogenous mouse constant region loci such that the mouse produces antibodies comprising a human heavy chain variable domain and a heavy chain constant region of mouse in response to antigenic stimulation.

Mice bearing a replacement of the endogenous mouse heavy chain variable region locus with the human heavy chain variable region locus and a single rearranged human light chain variable region substituted with histidine in the endogenous κ light chain locus are obtained. Inverse chimeric antibodies containing somatically mutated heavy chains (human heavy chain variable domain and mouse CH) are obtained with a single human light chain substituted with histidine (HULC, human light chain variable domain and mouse CL) after immunization with an antigen of interest. PH dependent human antibodies generated in said mice are identified using antibody isolation and scanning methods known in the art or described above. Nucleotide sequences of variable light and heavy chain regions of B cells expressing the antibodies, for example, pH sensitive antibodies, are identified and fully human antibodies are produced by fusion of the nucleotide sequences of heavy and light chain regions variables to C nucleotide sequences

Equivalent

 5

Those skilled in the art will recognize, or will be able to determine using nothing more than usual experimentation, many equivalents of the specific embodiments of the invention described herein.

SEQUENCE LIST 10

<110> Regeneron Pharmaceuticals, Inc.

<120> Light chain antibodies modified by genetic engineering with histidine and genetically modified non-human animals for their generation 15

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Claims (14)

1. A genetically modified rodent comprising in its germ line an immunoglobulin light chain locus comprising (i) a single rearranged gene region of human immunoglobulin light chain variable region comprising human VL and JL segment sequences and (ii ) an immunoglobulin light chain constant region gene sequence in operative binding to the unique human immunoglobulin light chain variable region rearranged gene sequence, wherein the only human region immunoglobulin light chain variable region rearranged gene sequence comprises at least one genetically engineered insert or replacement of a histidine codon that is not encoded by corresponding human germ line VL and JL gene sequences, 10 wherein the human immunoglobulin light chain rearranged variable region gene sequence alone encodes an immunoglobulin chain comprising a light chain variable domain, wherein the histidine codon is in the nucleotide sequence encoding a complementarity determining region (CDR) of the light chain variable domain, wherein the light chain variable domain comprises at least one histidine residue at an amino acid position encoded by the at least one inserted or substituted histidine codon, and wherein the immunoglobulin light chain encoded by the immunoglobulin light chain locus is capable of pairing with a plurality of immunoglobulin heavy chains selected by the rodent, in which each of the plurality of immunoglobulin heavy chains specifically bind at different epitopes when paired with the immunoglobulin light chain. twenty 2. The rodent of claim 1, wherein the plurality of immunoglobulin heavy chains comprises somatic mutations. 3. The rodent of claim 1, wherein: the immunoglobulin light chain constant region gene sequence is a non-human immunoglobulin light chain constant region gene sequence, such as an endogenous non-human immunoglobulin light chain constant region gene sequence, wherein the immunoglobulin light chain locus is optionally in an endogenous non-human immunoglobulin light chain locus. 4. The rodent of any one of the preceding claims, wherein: the rodent further comprises in its germ line an immunoglobulin heavy chain locus comprising a non-rearranged gene sequence of immunoglobulin heavy chain variable region comprising human VH, DH and JH segments operably linked to a constant region gene sequence of immunoglobulin heavy chain, optionally wherein the immunoglobulin heavy chain constant region gene sequence is a non-human immunoglobulin heavy chain constant region gene sequence, such as an endogenous non-human immunoglobulin constant region gene sequence, wherein the immunoglobulin heavy chain locus is optionally in an endogenous non-human immunoglobulin heavy chain locus. 5. The rodent of any one of the preceding claims, wherein the rodent lacks a variable region of functional non-rearranged immunoglobulin light chain. 6. The rodent of any one of the preceding claims, wherein the insertion or substitution engineered by histidine codon is in the nucleotide sequence encoding a complementarity determining region (CDR) 3, optionally wherein The histidine codon is one, two, three or 50 four CDR3 codons. 7. The rodent of any one of the preceding claims, wherein the only rearranged human immunoglobulin light chain variable region is derived from a human Vκ1-39 or Vκ3-20 gene segment, optionally in which the only variable chain region Lightly rearranged human immunoglobulin is derived from a gene sequence Vκ1-39 / Jκ5 or Vκ3-20 / Jκ1 rearranged. 8. The rodent of any one of the preceding claims, wherein the rodent comprises a population of B cells in response to an antigen of interest that is enriched for antibodies that exhibit a decrease in dissociative half-life (t1 / 2) at a Acid pH compared to a neutral pH of at least about 60 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times times, at least about 25 times, at least about 30 times or optionally about 30 times or more.  65 9. The rodent of any one of the preceding claims, wherein the rodent expresses an antibody comprising a human immunoglobulin light chain variable domain with a substitution of at least one non-histidine moiety with a histidine moiety in a position of amino acid encoded by the histidine codon modified by genetic engineering in the unique gene sequence rearrangement of immunoglobulin light chain variable region that is not encoded by V gene sequences 10. The rodent of any one of the preceding claims, wherein the single gene sequence reordered from human immunoglobulin light chain variable region replaces an unordered gene sequence from endogenous light chain variable region.  10 11. The rodent of any of the preceding claims, wherein the rodent is a rat. 12. The rodent of any one of claims 1 to 10, wherein the rodent is a mouse. 13. The rodent of any one of the preceding claims, wherein the single gene sequence reordered from human immunoglobulin light chain variable region is operably linked to a gene sequence of immunoglobulin light chain constant region selected from a gene sequence of rat or mouse immunoglobulin light chain constant region, wherein the immunoglobulin light chain locus is optionally in an endogenous mouse light chain locus; and 20 wherein the rodent optionally further comprises in its germ line an immunoglobulin heavy chain locus comprising an unordered sequence of immunoglobulin heavy chain variable region comprising human VH, DH and JH segments operably linked to a region gene sequence immunoglobulin heavy chain constant selected from a rat or mouse heavy chain constant region gene sequence; wherein the immunoglobulin heavy chain locus is optionally in an endogenous mouse immunoglobulin heavy chain locus. 14. The rodent of any one of the preceding claims, wherein the rodent expresses a population of antigen-specific antibodies in response to an antigen of interest in which all antibodies in the population comprise: immunoglobulin light chain variable domains derived from the same single human light chain variable region rearranged gene sequence comprising human VL and JL segment sequences comprising at least one genetically engineered histidine codon that is not encoded by gene sequences Corresponding human germ line VL and JL, and immunoglobulin heavy chains comprising human heavy chain variable domains derived from a repertoire of human heavy chain segments V, D and J.